Immunogenic proteins and compositions

ABSTRACT

The invention provides proteins and compositions for the treatment and prevention of Streptococcus agalactiae (Group B streptococcus; GBS).

This application, filed Jun. 22, 2017, is a continuation of U.S.application Ser. No. 15/252,930, filed Aug. 31, 2016, now U.S. Pat. No.9,725,488, issued Aug. 8, 2017, which is a continuation of U.S.application Ser. No. 14/631,456, filed Feb. 25, 2015, now U.S. Pat. No.9,458,229, issued Oct. 4, 2016, which is a continuation of U.S.application Ser. No. 13/637,929, filed Nov. 29, 2012, now U.S. Pat. No.9,079,946, issued Jul. 14, 2015, which was filed pursuant to 35 U.S.C. §371 as a U.S. National Stage Application of International PatentApplication Serial No. PCT/IB2011/051415, filed Apr. 1, 2011, whichclaims priority to U.K. Application Serial No. GB 1005625.7, filed Apr.1, 2010.

TECHNICAL FIELD

The invention provides proteins and compositions for the treatment andprevention of Streptococcus agalactiae (Group B streptococcus; GBS).

BACKGROUND ART

The Gram-positive bacterium Streptococcus agalactiae (or “group Bstreptococcus”, abbreviated to “GBS”) causes serious disease, bacteremiaand meningitis, in immunocompromised individuals and in neonates. Thereare two types of neonatal infection. The first (early onset, usuallywithin 5 days of birth) is manifested by bacteremia and pneumonia. It iscontracted vertically as a baby passes through the birth canal. GBScolonises the vagina of about 25% of young women, and approximately 1%of infants born via a vaginal birth to colonised mothers will becomeinfected. Mortality is between 50-70%. The second is a meningitis thatoccurs 10 to 60 days after birth. If pregnant women are vaccinated withtype III capsule so that the infants are passively immunised, theincidence of the late onset meningitis is reduced but is not entirelyeliminated.

The “B” in “GBS” refers to the Lancefield classification, which is basedon the antigenicity of a carbohydrate which is soluble in dilute acidand called the C carbohydrate. Lancefield identified 13 types of Ccarbohydrate, designated A to O, that could be serologicallydifferentiated. The organisms that most commonly infect humans are foundin groups A, B, D, and G. Within group B, strains can be divided into 10serotypes (Ia, Ib, II, III, IV, V, VI, VII, VIII and XI) based on thestructure of their polysaccharidecapsule.

Investigations have been conducted into the development of protein-basedand polysaccharide-based vaccines against GBS but currently, no GBSvaccine is commercially available. There therefore remains a need foreffective vaccines against S.agalactiae infection.

It is an object of the invention to provide proteins and immunogeniccompositions which can be used in the development of such vaccines.

DISCLOSURE OF THE INVENTION

Pilus structures in gram-positive bacteria are considered to beinteresting vaccine candidates. GBS has three pilus variants, eachencoded by a distinct pathogenicity island, PI-1, PI-2a and PI-2b [1,2]. Each pathogenicity island consists of 5 genes coding for: the pilusbackbone protein (BP); 2 ancillary proteins (AP1 and AP2); and 2 sortaseproteins that are involved in the assembly of the pili. All GBS strainscarry at least one of these 3 pathogenicity islands and the sequences ofthe pilus structural proteins (BP, AP1 and AP2) encoded by thesepathogenicity islands are generally well conserved. However, thesequence of the backbone protein encoded by pathogenicity island 2a(BP-2a), referred to herein as GBS59, varies between GBS strains. TheGBS59 pilus subunit has at least seven clades and the sequence identitybetween these clades is as low as 48%.

Reference amino acid sequences for the seven GBS59 clades are SEQ IDNO:1 (derived from GBS strain 2603), SEQ ID NO:2 (derived from GBSstrain 515), SEQ ID NO:3 (derived from GBS strain CJB111), SEQ ID NO:4(derived from GBS strain H36B), SEQ ID NO:5 (derived from GBS strainCJB110), SEQ ID NO:6 (derived from GBS strain DK21) and SEQ ID NO:7(derived from GBS strain NEM316) herein.

Serum raised against a given GBS59 clade is active against other strainsof GBS that express that clade, but is not active against strains whichexpress one of the other five clades, i.e. there is intra-cladecross-protection, but not inter-clade cross-protection.

According to the invention, therefore, an immunogenic composition isprovided which includes at least two different clades of GBS59. Thedifferent clades of GBS59 may be present in the immunogenic compositionas separate polypeptides or may be fused as a single polypeptide chain.The inclusion of multiple GBS59 clades as vaccine components improvesthe strain coverage of the immunogenic composition against GBS.

Furthermore, the inventors have identified domains within the GBS59clades containing epitopes responsible for inducing an immunogenicresponse. The immunogenic composition may therefore include fragments ofat least two different clades of GBS59 comprising one or more of thesedomains, or sub-fragments of these domains, instead of the full-lengthGBS59 proteins. Alternatively, these fragments of at least two differentclades of GBS59 may be fused as a single polypeptide chain. The use offragments of GBS59 clades in place of full-length proteins facilitatesthe preparation of a vaccine with improved strain coverage against GBS.

Thus, the invention provides an immunogenic composition comprising atleast two of:

-   -   a) a first polypeptide comprising a first amino acid sequence,        wherein the first amino acid sequence comprises an amino acid        sequence (i) having at least a% sequence identity to SEQ ID NO:1        and/or (ii) consisting of a fragment of at least t contiguous        amino acids from SEQ ID NO:1 or from a sequence having at least        a% sequence identity to SEQ ID NO:1;    -   b) a second polypeptide comprising a second amino acid sequence,        wherein the second amino acid sequence comprises an amino acid        sequence (i) having at least b% sequence identity to SEQ ID NO:2        and/or (ii) consisting of a fragment of at least u contiguous        amino acids from SEQ ID NO:2 or from a sequence having at least        b% sequence identity to SEQ ID NO:1;    -   c) a third polypeptide comprising a third amino acid sequence,        wherein the third amino acid sequence comprises an amino acid        sequence (i) having at least c% sequence identity to SEQ ID NO:3        and/or (ii) consisting of a fragment of at least v contiguous        amino acids from SEQ ID NO:3 or from a sequence having at least        c% sequence identity to SEQ ID NO:3;    -   d) a fourth polypeptide comprising a fourth amino acid sequence,        wherein the fourth amino acid sequence comprises an amino acid        sequence (i) having at least d% sequence identity to SEQ ID NO:4        and/or (ii) consisting of a fragment of at least w contiguous        amino acids from SEQ ID NO:4 or from a sequence having at least        d% sequence identity to SEQ ID NO:4;    -   e) a fifth polypeptide comprising a fifth amino acid sequence,        wherein the fifth amino acid sequence comprises an amino acid        sequence (i) having at least e% sequence identity to SEQ ID NO:5        and/or (ii) consisting of a fragment of at least x contiguous        amino acids from SEQ ID NO:5 or from a sequence having at least        e% sequence identity to SEQ ID NO:5; and/or    -   f) a sixth polypeptide comprising a sixth amino acid sequence,        wherein the sixth amino acid sequence comprises an amino acid        sequence (i) having at least f% sequence identity to SEQ ID NO:6        and/or (ii) consisting of a fragment of at least y contiguous        amino acids from SEQ ID NO:6 or from a sequence having at least        f% sequence identity to SEQ ID NO:6; and/or    -   g) a seventh polypeptide comprising a seventh amino acid        sequence, wherein the seventh amino acid sequence comprises an        amino acid sequence (i) having at least g% sequence identity to        SEQ ID NO:7 and/or (ii) consisting of a fragment of at least z        contiguous amino acids from SEQ ID NO:7 or from a sequence        having at least g% sequence identity to SEQ ID NO:7.

The immunogenic composition may comprise 2, 3, 4, 5, 6 or all 7 of theseven amino acid sequences.

The invention also provides a polypeptide comprising at least two of:

-   -   a) a first polypeptide comprising a first amino acid sequence,        wherein the first amino acid sequence comprises an amino acid        sequence (i) having at least a% sequence identity to SEQ ID NO:1        and/or (ii) consisting of a fragment of at least t contiguous        amino acids from SEQ ID NO:1 or from a sequence having at least        a% sequence identity to SEQ ID NO:1;    -   b) a second polypeptide comprising a second amino acid sequence,        wherein the second amino acid sequence comprises an amino acid        sequence (i) having at least b% sequence identity to SEQ ID NO:2        and/or (ii) consisting of a fragment of at least u contiguous        amino acids from SEQ ID NO:2 or from a sequence having at least        b% sequence identity to SEQ ID NO:1;    -   c) a third polypeptide comprising a third amino acid sequence,        wherein the third amino acid sequence comprises an amino acid        sequence (i) having at least c% sequence identity to SEQ ID NO:3        and/or (ii) consisting of a fragment of at least v contiguous        amino acids from SEQ ID NO:3 or from a sequence having at least        c% sequence identity to SEQ ID NO:3;    -   d) a fourth polypeptide comprising a fourth amino acid sequence,        wherein the fourth amino acid sequence comprises an amino acid        sequence (i) having at least d% sequence identity to SEQ ID NO:4        and/or (ii) consisting of a fragment of at least w contiguous        amino acids from SEQ ID NO:4 or from a sequence having at least        d% sequence identity to SEQ ID NO:4;    -   e) a fifth polypeptide comprising a fifth amino acid sequence,        wherein the fifth amino acid sequence comprises an amino acid        sequence (i) having at least e% sequence identity to SEQ ID NO:5        and/or (ii) consisting of a fragment of at least x contiguous        amino acids from SEQ ID NO:5 or from a sequence having at least        e% sequence identity to SEQ ID NO:5; and/or    -   f) a sixth polypeptide comprising a sixth amino acid sequence,        wherein the sixth amino acid sequence comprises an amino acid        sequence (i) having at least f% sequence identity to SEQ ID NO:6        and/or (ii) consisting of a fragment of at least y contiguous        amino acids from SEQ ID NO:6 or from a sequence having at least        f% sequence identity to SEQ ID NO:6; and/or    -   g) a seventh polypeptide comprising a seventh amino acid        sequence, wherein the seventh amino acid sequence comprises an        amino acid sequence (i) having at least g% sequence identity to        SEQ ID NO:7 and/or (ii) consisting of a fragment of at least z        contiguous amino acids from SEQ ID NO:7 or from a sequence        having at least g% sequence identity to SEQ ID NO:7.

The polypeptide may comprise 2, 3, 4, 5, 6 or all 7 of the seven aminoacid sequences.

The invention also provides a polypeptide comprising amino acidsequence:-A-{-X-L-}_(n)—B—

wherein: X is an amino acid sequence of a first polypeptide, secondpolypeptide, third polypeptide, fourth polypeptide, fifth polypeptide,sixth polypeptide or seventh polypeptide as defined above; L is anoptional linker amino acid sequence; A is an optional N-terminal aminoacid sequence; B is an optional C-terminal amino acid sequence; n is aninteger or 2 or more. Typically, n is 2, 3, 4, 5, 6 or 7. The X moietiesin the polypeptide are different, as discussed below.

Where n is 2, X moieties are selected from the following:

n X₁ X₂ 2 First polypeptide Second polypeptide 2 Second polypeptideFirst polypeptide 2 First polypeptide Third polypeptide 2 Thirdpolypeptide First polypeptide 2 First polypeptide Fourth polypeptide 2Fourth polypeptide First polypeptide 2 Fifth polypeptide Firstpolypeptide 2 First polypeptide Sixth polypeptide 2 Sixth polypeptideFirst polypeptide 2 Second polypeptide Third polypeptide 2 Thirdpolypeptide Second polypeptide 2 Second polypeptide Fourth polypeptide 2Fourth polypeptide Second polypeptide 2 Second polypeptide Fifthpolypeptide 2 Fifth polypeptide Second polypeptide 2 Second polypeptideSixth polypeptide 2 Sixth polypeptide Second polypeptide 2 Thirdpolypeptide Fourth polypeptide 2 Fourth polypeptide Third polypeptide 2Third polypeptide Fifth polypeptide 2 Fifth polypeptide Thirdpolypeptide 2 Third polypeptide Sixth polypeptide 2 Sixth polypeptideThird polypeptide 2 Fourth polypeptide Fifth polypeptide 2 Fifthpolypeptide Fourth polypeptide 2 Fourth polypeptide e Sixth polypeptide2 Sixth polypeptide Fourth polypeptide 2 Fifth polypeptide Sixthpolypeptide 2 Sixth polypeptide Fifth polypeptide

Where n is 3, X moieties are selected from the following:

n X₁ X₂ X₃ 3 First Polypeptide Second Polypeptide Third Polypeptide 3First Polypeptide Second Polypeptide Fourth Polypeptide 3 FirstPolypeptide Second Polypeptide Fifth Polypeptide 3 First PolypeptideSecond Polypeptide Sixth Polypeptide 3 First Polypeptide ThirdPolypeptide Second Polypeptide 3 First Polypeptide Third PolypeptideFourth Polypeptide 3 First Polypeptide Third Polypeptide FifthPolypeptide 3 First Polypeptide Third Polypeptide Sixth Polypeptide 3First Polypeptide Fourth Polypeptide Second Polypeptide 3 FirstPolypeptide Fourth Polypeptide Third Polypeptide 3 First PolypeptideFourth Polypeptide Fifth Polypeptide 3 First Polypeptide FourthPolypeptide Sixth Polypeptide 3 First Polypeptide Fifth PolypeptideSecond Polypeptide 3 First Polypeptide Fifth Polypeptide ThirdPolypeptide 3 First Polypeptide Fifth Polypeptide Fourth Polypeptide 3First Polypeptide Fifth Polypeptide Sixth Polypeptide 3 FirstPolypeptide Sixth Polypeptide Second Polypeptide 3 First PolypeptideSixth Polypeptide Third Polypeptide 3 First Polypeptide SixthPolypeptide Fourth Polypeptide 3 First Polypeptide Sixth PolypeptideFifth Polypeptide 3 Second Polypeptide First Polypeptide ThirdPolypeptide 3 Second Polypeptide First Polypeptide Fourth Polypeptide 3Second Polypeptide First Polypeptide Fifth Polypeptide 3 SecondPolypeptide First Polypeptide Sixth Polypeptide 3 Second PolypeptideThird Polypeptide First Polypeptide 3 Second Polypeptide ThirdPolypeptide Fourth Polypeptide 3 Second Polypeptide Third PolypeptideFifth Polypeptide 3 Second Polypeptide Third Polypeptide SixthPolypeptide 3 Second Polypeptide Fourth Polypeptide First Polypeptide 3Second Polypeptide Fourth Polypeptide Third Polypeptide 3 SecondPolypeptide Fourth Polypeptide Fifth Polypeptide 3 Second PolypeptideFourth Polypeptide Sixth Polypeptide 3 Second Polypeptide FifthPolypeptide First Polypeptide 3 Second Polypeptide Fifth PolypeptideThird Polypeptide 3 Second Polypeptide Fifth Polypeptide FourthPolypeptide 3 Second Polypeptide Fifth Polypeptide Sixth Polypeptide 3Second Polypeptide Sixth Polypeptide First Polypeptide 3 SecondPolypeptide Sixth Polypeptide Third Polypeptide 3 Second PolypeptideSixth Polypeptide Fourth Polypeptide 3 Second Polypeptide SixthPolypeptide Fifth Polypeptide 3 Third Polypeptide First PolypeptideSecond Polypeptide 3 Third Polypeptide First Polypeptide FourthPolypeptide 3 Third Polypeptide First Polypeptide Fifth Polypeptide 3Third Polypeptide First Polypeptide Sixth Polypeptide 3 ThirdPolypeptide Second Polypeptide First Polypeptide 3 Third PolypeptideSecond Polypeptide Fourth Polypeptide 3 Third Polypeptide SecondPolypeptide Fifth Polypeptide 3 Third Polypeptide Second PolypeptideSixth Polypeptide 3 Third Polypeptide Fourth Polypeptide FirstPolypeptide 3 Third Polypeptide Fourth Polypeptide Second Polypeptide 3Third Polypeptide Fourth Polypeptide Fifth Polypeptide 3 ThirdPolypeptide Fourth Polypeptide Sixth Polypeptide 3 Third PolypeptideFifth Polypeptide First Polypeptide 3 Third Polypeptide FifthPolypeptide Second Polypeptide 3 Third Polypeptide Fifth PolypeptideFourth Polypeptide 3 Third Polypeptide Fifth Polypeptide SixthPolypeptide 3 Third Polypeptide Sixth Polypeptide First Polypeptide 3Third Polypeptide Sixth Polypeptide Second Polypeptide 3 ThirdPolypeptide Sixth Polypeptide Fourth Polypeptide 3 Third PolypeptideSixth Polypeptide Fifth Polypeptide 3 Fourth Polypeptide FirstPolypeptide Second Polypeptide 3 Fourth Polypeptide First PolypeptideThird Polypeptide 3 Fourth Polypeptide First Polypeptide FifthPolypeptide 3 Fourth Polypeptide First Polypeptide Sixth Polypeptide 3Fourth Polypeptide Second Polypeptide First Polypeptide 3 FourthPolypeptide Second Polypeptide Third Polypeptide 3 Fourth PolypeptideSecond Polypeptide Fifth Polypeptide 3 Fourth Polypeptide SecondPolypeptide Sixth Polypeptide 3 Fourth Polypeptide Third PolypeptideFirst Polypeptide 3 Fourth Polypeptide Third Polypeptide SecondPolypeptide 3 Fourth Polypeptide Third Polypeptide Fifth Polypeptide 3Fourth Polypeptide Third Polypeptide Sixth Polypeptide 3 FourthPolypeptide Fifth Polypeptide First Polypeptide 3 Fourth PolypeptideFifth Polypeptide Second Polypeptide 3 Fourth Polypeptide FifthPolypeptide Third Polypeptide 3 Fourth Polypeptide Fifth PolypeptideSixth Polypeptide 3 Fourth Polypeptide Sixth Polypeptide FirstPolypeptide 3 Fourth Polypeptide Sixth Polypeptide Second Polypeptide 3Fourth Polypeptide Sixth Polypeptide Third Polypeptide 3 FourthPolypeptide Sixth Polypeptide Fifth Polypeptide 3 Fifth PolypeptideFirst Polypeptide Second Polypeptide 3 Fifth Polypeptide FirstPolypeptide Third Polypeptide 3 Fifth Polypeptide First PolypeptideFourth Polypeptide 3 Fifth Polypeptide First Polypeptide SixthPolypeptide 3 Fifth Polypeptide Second Polypeptide First Polypeptide 3Fifth Polypeptide Second Polypeptide Third Polypeptide 3 FifthPolypeptide Second Polypeptide Fourth Polypeptide 3 Fifth PolypeptideSecond Polypeptide Sixth Polypeptide 3 Fifth Polypeptide ThirdPolypeptide First Polypeptide 3 Fifth Polypeptide Third PolypeptideSecond Polypeptide 3 Fifth Polypeptide Third Polypeptide FourthPolypeptide 3 Fifth Polypeptide Third Polypeptide Sixth Polypeptide 3Fifth Polypeptide Fourth Polypeptide First Polypeptide 3 FifthPolypeptide Fourth Polypeptide Second Polypeptide 3 Fifth PolypeptideFourth Polypeptide Third Polypeptide 3 Fifth Polypeptide FourthPolypeptide Sixth Polypeptide 3 Fifth Polypeptide Sixth PolypeptideFirst Polypeptide 3 Fifth Polypeptide Sixth Polypeptide SecondPolypeptide 3 Fifth Polypeptide Sixth Polypeptide Third Polypeptide 3Fifth Polypeptide Sixth Polypeptide Fourth Polypeptide 3 SixthPolypeptide First Polypeptide Second Polypeptide 3 Sixth PolypeptideFirst Polypeptide Third Polypeptide 3 Sixth Polypeptide FirstPolypeptide Fourth Polypeptide 3 Sixth Polypeptide First PolypeptideFifth Polypeptide 3 Sixth Polypeptide Second Polypeptide FirstPolypeptide 3 Sixth Polypeptide Second Polypeptide Third Polypeptide 3Sixth Polypeptide Second Polypeptide Fourth Polypeptide 3 SixthPolypeptide Second Polypeptide Fifth Polypeptide 3 Sixth PolypeptideThird Polypeptide First Polypeptide 3 Sixth Polypeptide ThirdPolypeptide Second Polypeptide 3 Sixth Polypeptide Third PolypeptideFourth Polypeptide 3 Sixth Polypeptide Third Polypeptide FifthPolypeptide 3 Sixth Polypeptide Fourth Polypeptide First Polypeptide 3Sixth Polypeptide Fourth Polypeptide Second Polypeptide 3 SixthPolypeptide Fourth Polypeptide Third Polypeptide 3 Sixth PolypeptideFourth Polypeptide Fifth Polypeptide 3 Sixth Polypeptide FifthPolypeptide First Polypeptide 3 Sixth Polypeptide Fifth PolypeptideSecond Polypeptide 3 Sixth Polypeptide Fifth Polypeptide ThirdPolypeptide 3 Sixth Polypeptide Fifth Polypeptide Fourth Polypeptide

Where n is 4, X moieties are selected from the following:

n X₁ X₂ X₃ X₄ 4 First Polypeptide Second Polypeptide Third PolypeptideFourth Polypeptide 4 First Polypeptide Second Polypeptide ThirdPolypeptide Fifth Polypeptide 4 First Polypeptide Second PolypeptideThird Polypeptide Sixth Polypeptide 4 First Polypeptide SecondPolypeptide Fourth Polypeptide Third Polypeptide 4 First PolypeptideSecond Polypeptide Fourth Polypeptide Fifth Polypeptide 4 FirstPolypeptide Second Polypeptide Fourth Polypeptide Sixth Polypeptide 4First Polypeptide Second Polypeptide Fifth Polypeptide Third Polypeptide4 First Polypeptide Second Polypeptide Fifth Polypeptide FourthPolypeptide 4 First Polypeptide Second Polypeptide Fifth PolypeptideSixth Polypeptide 4 First Polypeptide Second Polypeptide SixthPolypeptide Third Polypeptide 4 First Polypeptide Second PolypeptideSixth Polypeptide Fourth Polypeptide 4 First Polypeptide SecondPolypeptide Sixth Polypeptide Fifth Polypeptide 4 First PolypeptideThird Polypeptide Second Polypeptide Fourth Polypeptide 4 FirstPolypeptide Third Polypeptide Second Polypeptide Fifth Polypeptide 4First Polypeptide Third Polypeptide Second Polypeptide Sixth Polypeptide4 First Polypeptide Third Polypeptide Fourth Polypeptide SecondPolypeptide 4 First Polypeptide Third Polypeptide Fourth PolypeptideFifth Polypeptide 4 First Polypeptide Third Polypeptide FourthPolypeptide Sixth Polypeptide 4 First Polypeptide Third PolypeptideFifth Polypeptide Second Polypeptide 4 First Polypeptide ThirdPolypeptide Fifth Polypeptide Fourth Polypeptide 4 First PolypeptideThird Polypeptide Fifth Polypeptide Sixth Polypeptide 4 FirstPolypeptide Third Polypeptide Sixth Polypeptide Second Polypeptide 4First Polypeptide Third Polypeptide Sixth Polypeptide Fourth Polypeptide4 First Polypeptide Third Polypeptide Sixth Polypeptide FifthPolypeptide 4 First Polypeptide Fourth Polypeptide Second PolypeptideThird Polypeptide 4 First Polypeptide Fourth Polypeptide SecondPolypeptide Fifth Polypeptide 4 First Polypeptide Fourth PolypeptideSecond Polypeptide Sixth Polypeptide 4 First Polypeptide FourthPolypeptide Third Polypeptide Second Polypeptide 4 First PolypeptideFourth Polypeptide Third Polypeptide Fifth Polypeptide 4 FirstPolypeptide Fourth Polypeptide Third Polypeptide Sixth Polypeptide 4First Polypeptide Fourth Polypeptide Fifth Polypeptide SecondPolypeptide 4 First Polypeptide Fourth Polypeptide Fifth PolypeptideThird Polypeptide 4 First Polypeptide Fourth Polypeptide FifthPolypeptide Sixth Polypeptide 4 First Polypeptide Fourth PolypeptideSixth Polypeptide Second Polypeptide 4 First Polypeptide FourthPolypeptide Sixth Polypeptide Third Polypeptide 4 First PolypeptideFourth Polypeptide Sixth Polypeptide Fifth Polypeptide 4 FirstPolypeptide Fifth Polypeptide Second Polypeptide Third Polypeptide 4First Polypeptide Fifth Polypeptide Second Polypeptide FourthPolypeptide 4 First Polypeptide Fifth Polypeptide Second PolypeptideSixth Polypeptide 4 First Polypeptide Fifth Polypeptide ThirdPolypeptide Second Polypeptide 4 First Polypeptide Fifth PolypeptideThird Polypeptide Fourth Polypeptide 4 First Polypeptide FifthPolypeptide Third Polypeptide Sixth Polypeptide 4 First PolypeptideFifth Polypeptide Fourth Polypeptide Second Polypeptide 4 FirstPolypeptide Fifth Polypeptide Fourth Polypeptide Third Polypeptide 4First Polypeptide Fifth Polypeptide Fourth Polypeptide Sixth Polypeptide4 First Polypeptide Fifth Polypeptide Sixth Polypeptide SecondPolypeptide 4 First Polypeptide Fifth Polypeptide Sixth PolypeptideThird Polypeptide 4 First Polypeptide Fifth Polypeptide SixthPolypeptide Fourth Polypeptide 4 First Polypeptide Sixth PolypeptideSecond Polypeptide Third Polypeptide 4 First Polypeptide SixthPolypeptide Second Polypeptide Fourth Polypeptide 4 First PolypeptideSixth Polypeptide Second Polypeptide Fifth Polypeptide 4 FirstPolypeptide Sixth Polypeptide Third Polypeptide Second Polypeptide 4First Polypeptide Sixth Polypeptide Third Polypeptide Fourth Polypeptide4 First Polypeptide Sixth Polypeptide Third Polypeptide FifthPolypeptide 4 First Polypeptide Sixth Polypeptide Fourth PolypeptideSecond Polypeptide 4 First Polypeptide Sixth Polypeptide FourthPolypeptide Third Polypeptide 4 First Polypeptide Sixth PolypeptideFourth Polypeptide Fifth Polypeptide 4 First Polypeptide SixthPolypeptide Fifth Polypeptide Second Polypeptide 4 First PolypeptideSixth Polypeptide Fifth Polypeptide Third Polypeptide 4 FirstPolypeptide Sixth Polypeptide Fifth Polypeptide Fourth Polypeptide 4Second Polypeptide First Polypeptide Third Polypeptide FourthPolypeptide 4 Second Polypeptide First Polypeptide Third PolypeptideFifth Polypeptide 4 Second Polypeptide First Polypeptide ThirdPolypeptide Sixth Polypeptide 4 Second Polypeptide First PolypeptideFourth Polypeptide Third Polypeptide 4 Second Polypeptide FirstPolypeptide Fourth Polypeptide Fifth Polypeptide 4 Second PolypeptideFirst Polypeptide Fourth Polypeptide Sixth Polypeptide 4 SecondPolypeptide First Polypeptide Fifth Polypeptide Third Polypeptide 4Second Polypeptide First Polypeptide Fifth Polypeptide FourthPolypeptide 4 Second Polypeptide First Polypeptide Fifth PolypeptideSixth Polypeptide 4 Second Polypeptide First Polypeptide SixthPolypeptide Third Polypeptide 4 Second Polypeptide First PolypeptideSixth Polypeptide Fourth Polypeptide 4 Second Polypeptide FirstPolypeptide Sixth Polypeptide Fifth Polypeptide 4 Second PolypeptideThird Polypeptide First Polypeptide Fourth Polypeptide 4 SecondPolypeptide Third Polypeptide First Polypeptide Fifth Polypeptide 4Second Polypeptide Third Polypeptide First Polypeptide Sixth Polypeptide4 Second Polypeptide Third Polypeptide Fourth Polypeptide FirstPolypeptide 4 Second Polypeptide Third Polypeptide Fourth PolypeptideFifth Polypeptide 4 Second Polypeptide Third Polypeptide FourthPolypeptide Sixth Polypeptide 4 Second Polypeptide Third PolypeptideFifth Polypeptide First Polypeptide 4 Second Polypeptide ThirdPolypeptide Fifth Polypeptide Fourth Polypeptide 4 Second PolypeptideThird Polypeptide Fifth Polypeptide Sixth Polypeptide 4 SecondPolypeptide Third Polypeptide Sixth Polypeptide First Polypeptide 4Second Polypeptide Third Polypeptide Sixth Polypeptide FourthPolypeptide 4 Second Polypeptide Third Polypeptide Sixth PolypeptideFifth Polypeptide 4 Second Polypeptide Fourth Polypeptide FirstPolypeptide Third Polypeptide 4 Second Polypeptide Fourth PolypeptideFirst Polypeptide Fifth Polypeptide 4 Second Polypeptide FourthPolypeptide First Polypeptide Sixth Polypeptide 4 Second PolypeptideFourth Polypeptide Third Polypeptide First Polypeptide 4 SecondPolypeptide Fourth Polypeptide Third Polypeptide Fifth Polypeptide 4Second Polypeptide Fourth Polypeptide Third Polypeptide SixthPolypeptide 4 Second Polypeptide Fourth Polypeptide Fifth PolypeptideFirst Polypeptide 4 Second Polypeptide Fourth Polypeptide FifthPolypeptide Third Polypeptide 4 Second Polypeptide Fourth PolypeptideFifth Polypeptide Sixth Polypeptide 4 Second Polypeptide FourthPolypeptide Sixth Polypeptide First Polypeptide 4 Second PolypeptideFourth Polypeptide Sixth Polypeptide Third Polypeptide 4 SecondPolypeptide Fourth Polypeptide Sixth Polypeptide Fifth Polypeptide 4Second Polypeptide Fifth Polypeptide First Polypeptide Third Polypeptide4 Second Polypeptide Fifth Polypeptide First Polypeptide FourthPolypeptide 4 Second Polypeptide Fifth Polypeptide First PolypeptideSixth Polypeptide 4 Second Polypeptide Fifth Polypeptide ThirdPolypeptide First Polypeptide 4 Second Polypeptide Fifth PolypeptideThird Polypeptide Fourth Polypeptide 4 Second Polypeptide FifthPolypeptide Third Polypeptide Sixth Polypeptide 4 Second PolypeptideFifth Polypeptide Fourth Polypeptide First Polypeptide 4 SecondPolypeptide Fifth Polypeptide Fourth Polypeptide Third Polypeptide 4Second Polypeptide Fifth Polypeptide Fourth Polypeptide SixthPolypeptide 4 Second Polypeptide Fifth Polypeptide Sixth PolypeptideFirst Polypeptide 4 Second Polypeptide Fifth Polypeptide SixthPolypeptide Third Polypeptide 4 Second Polypeptide Fifth PolypeptideSixth Polypeptide Fourth Polypeptide 4 Second Polypeptide SixthPolypeptide First Polypeptide Third Polypeptide 4 Second PolypeptideSixth Polypeptide First Polypeptide Fourth Polypeptide 4 SecondPolypeptide Sixth Polypeptide First Polypeptide Fifth Polypeptide 4Second Polypeptide Sixth Polypeptide Third Polypeptide First Polypeptide4 Second Polypeptide Sixth Polypeptide Third Polypeptide FourthPolypeptide 4 Second Polypeptide Sixth Polypeptide Third PolypeptideFifth Polypeptide 4 Second Polypeptide Sixth Polypeptide FourthPolypeptide First Polypeptide 4 Second Polypeptide Sixth PolypeptideFourth Polypeptide Third Polypeptide 4 Second Polypeptide SixthPolypeptide Fourth Polypeptide Fifth Polypeptide 4 Second PolypeptideSixth Polypeptide Fifth Polypeptide First Polypeptide 4 SecondPolypeptide Sixth Polypeptide Fifth Polypeptide Third Polypeptide 4Second Polypeptide Sixth Polypeptide Fifth Polypeptide FourthPolypeptide 4 Third Polypeptide First Polypeptide Second PolypeptideFourth Polypeptide 4 Third Polypeptide First Polypeptide SecondPolypeptide Fifth Polypeptide 4 Third Polypeptide First PolypeptideSecond Polypeptide Sixth Polypeptide 4 Third Polypeptide FirstPolypeptide Fourth Polypeptide Second Polypeptide 4 Third PolypeptideFirst Polypeptide Fourth Polypeptide Fifth Polypeptide 4 ThirdPolypeptide First Polypeptide Fourth Polypeptide Sixth Polypeptide 4Third Polypeptide First Polypeptide Fifth Polypeptide Second Polypeptide4 Third Polypeptide First Polypeptide Fifth Polypeptide FourthPolypeptide 4 Third Polypeptide First Polypeptide Fifth PolypeptideSixth Polypeptide 4 Third Polypeptide First Polypeptide SixthPolypeptide Second Polypeptide 4 Third Polypeptide First PolypeptideSixth Polypeptide Fourth Polypeptide 4 Third Polypeptide FirstPolypeptide Sixth Polypeptide Fifth Polypeptide 4 Third PolypeptideSecond Polypeptide First Polypeptide Fourth Polypeptide 4 ThirdPolypeptide Second Polypeptide First Polypeptide Fifth Polypeptide 4Third Polypeptide Second Polypeptide First Polypeptide Sixth Polypeptide4 Third Polypeptide Second Polypeptide Fourth Polypeptide FirstPolypeptide 4 Third Polypeptide Second Polypeptide Fourth PolypeptideFifth Polypeptide 4 Third Polypeptide Second Polypeptide FourthPolypeptide Sixth Polypeptide 4 Third Polypeptide Second PolypeptideFifth Polypeptide First Polypeptide 4 Third Polypeptide SecondPolypeptide Fifth Polypeptide Fourth Polypeptide 4 Third PolypeptideSecond Polypeptide Fifth Polypeptide Sixth Polypeptide 4 ThirdPolypeptide Second Polypeptide Sixth Polypeptide First Polypeptide 4Third Polypeptide Second Polypeptide Sixth Polypeptide FourthPolypeptide 4 Third Polypeptide Second Polypeptide Sixth PolypeptideFifth Polypeptide 4 Third Polypeptide Fourth Polypeptide FirstPolypeptide Second Polypeptide 4 Third Polypeptide Fourth PolypeptideFirst Polypeptide Fifth Polypeptide 4 Third Polypeptide FourthPolypeptide First Polypeptide Sixth Polypeptide 4 Third PolypeptideFourth Polypeptide Second Polypeptide First Polypeptide 4 ThirdPolypeptide Fourth Polypeptide Second Polypeptide Fifth Polypeptide 4Third Polypeptide Fourth Polypeptide Second Polypeptide SixthPolypeptide 4 Third Polypeptide Fourth Polypeptide Fifth PolypeptideFirst Polypeptide 4 Third Polypeptide Fourth Polypeptide FifthPolypeptide Second Polypeptide 4 Third Polypeptide Fourth PolypeptideFifth Polypeptide Sixth Polypeptide 4 Third Polypeptide FourthPolypeptide Sixth Polypeptide First Polypeptide 4 Third PolypeptideFourth Polypeptide Sixth Polypeptide Second Polypeptide 4 ThirdPolypeptide Fourth Polypeptide Sixth Polypeptide Fifth Polypeptide 4Third Polypeptide Fifth Polypeptide First Polypeptide Second Polypeptide4 Third Polypeptide Fifth Polypeptide First Polypeptide FourthPolypeptide 4 Third Polypeptide Fifth Polypeptide First PolypeptideSixth Polypeptide 4 Third Polypeptide Fifth Polypeptide SecondPolypeptide First Polypeptide 4 Third Polypeptide Fifth PolypeptideSecond Polypeptide Fourth Polypeptide 4 Third Polypeptide FifthPolypeptide Second Polypeptide Sixth Polypeptide 4 Third PolypeptideFifth Polypeptide Fourth Polypeptide First Polypeptide 4 ThirdPolypeptide Fifth Polypeptide Fourth Polypeptide Second Polypeptide 4Third Polypeptide Fifth Polypeptide Fourth Polypeptide Sixth Polypeptide4 Third Polypeptide Fifth Polypeptide Sixth Polypeptide FirstPolypeptide 4 Third Polypeptide Fifth Polypeptide Sixth PolypeptideSecond Polypeptide 4 Third Polypeptide Fifth Polypeptide SixthPolypeptide Fourth Polypeptide 4 Third Polypeptide Sixth PolypeptideFirst Polypeptide Second Polypeptide 4 Third Polypeptide SixthPolypeptide First Polypeptide Fourth Polypeptide 4 Third PolypeptideSixth Polypeptide First Polypeptide Fifth Polypeptide 4 ThirdPolypeptide Sixth Polypeptide Second Polypeptide First Polypeptide 4Third Polypeptide Sixth Polypeptide Second Polypeptide FourthPolypeptide 4 Third Polypeptide Sixth Polypeptide Second PolypeptideFifth Polypeptide 4 Third Polypeptide Sixth Polypeptide FourthPolypeptide First Polypeptide 4 Third Polypeptide Sixth PolypeptideFourth Polypeptide Second Polypeptide 4 Third Polypeptide SixthPolypeptide Fourth Polypeptide Fifth Polypeptide 4 Third PolypeptideSixth Polypeptide Fifth Polypeptide First Polypeptide 4 ThirdPolypeptide Sixth Polypeptide Fifth Polypeptide Second Polypeptide 4Third Polypeptide Sixth Polypeptide Fifth Polypeptide Fourth Polypeptide4 Fourth Polypeptide First Polypeptide Second Polypeptide ThirdPolypeptide 4 Fourth Polypeptide First Polypeptide Second PolypeptideFifth Polypeptide 4 Fourth Polypeptide First Polypeptide SecondPolypeptide Sixth Polypeptide 4 Fourth Polypeptide First PolypeptideThird Polypeptide Second Polypeptide 4 Fourth Polypeptide FirstPolypeptide Third Polypeptide Fifth Polypeptide 4 Fourth PolypeptideFirst Polypeptide Third Polypeptide Sixth Polypeptide 4 FourthPolypeptide First Polypeptide Fifth Polypeptide Second Polypeptide 4Fourth Polypeptide First Polypeptide Fifth Polypeptide Third Polypeptide4 Fourth Polypeptide First Polypeptide Fifth Polypeptide SixthPolypeptide 4 Fourth Polypeptide First Polypeptide Sixth PolypeptideSecond Polypeptide 4 Fourth Polypeptide First Polypeptide SixthPolypeptide Third Polypeptide 4 Fourth Polypeptide First PolypeptideSixth Polypeptide Fifth Polypeptide 4 Fourth Polypeptide SecondPolypeptide First Polypeptide Third Polypeptide 4 Fourth PolypeptideSecond Polypeptide First Polypeptide Fifth Polypeptide 4 FourthPolypeptide Second Polypeptide First Polypeptide Sixth Polypeptide 4Fourth Polypeptide Second Polypeptide Third Polypeptide FirstPolypeptide 4 Fourth Polypeptide Second Polypeptide Third PolypeptideFifth Polypeptide 4 Fourth Polypeptide Second Polypeptide ThirdPolypeptide Sixth Polypeptide 4 Fourth Polypeptide Second PolypeptideFifth Polypeptide First Polypeptide 4 Fourth Polypeptide SecondPolypeptide Fifth Polypeptide Third Polypeptide 4 Fourth PolypeptideSecond Polypeptide Fifth Polypeptide Sixth Polypeptide 4 FourthPolypeptide Second Polypeptide Sixth Polypeptide First Polypeptide 4Fourth Polypeptide Second Polypeptide Sixth Polypeptide ThirdPolypeptide 4 Fourth Polypeptide Second Polypeptide Sixth PolypeptideFifth Polypeptide 4 Fourth Polypeptide Third Polypeptide FirstPolypeptide Second Polypeptide 4 Fourth Polypeptide Third PolypeptideFirst Polypeptide Fifth Polypeptide 4 Fourth Polypeptide ThirdPolypeptide First Polypeptide Sixth Polypeptide 4 Fourth PolypeptideThird Polypeptide Second Polypeptide First Polypeptide 4 FourthPolypeptide Third Polypeptide Second Polypeptide Fifth Polypeptide 4Fourth Polypeptide Third Polypeptide Second Polypeptide SixthPolypeptide 4 Fourth Polypeptide Third Polypeptide Fifth PolypeptideFirst Polypeptide 4 Fourth Polypeptide Third Polypeptide FifthPolypeptide Second Polypeptide 4 Fourth Polypeptide Third PolypeptideFifth Polypeptide Sixth Polypeptide 4 Fourth Polypeptide ThirdPolypeptide Sixth Polypeptide First Polypeptide 4 Fourth PolypeptideThird Polypeptide Sixth Polypeptide Second Polypeptide 4 FourthPolypeptide Third Polypeptide Sixth Polypeptide Fifth Polypeptide 4Fourth Polypeptide Fifth Polypeptide First Polypeptide SecondPolypeptide 4 Fourth Polypeptide Fifth Polypeptide First PolypeptideThird Polypeptide 4 Fourth Polypeptide Fifth Polypeptide FirstPolypeptide Sixth Polypeptide 4 Fourth Polypeptide Fifth PolypeptideSecond Polypeptide First Polypeptide 4 Fourth Polypeptide FifthPolypeptide Second Polypeptide Third Polypeptide 4 Fourth PolypeptideFifth Polypeptide Second Polypeptide Sixth Polypeptide 4 FourthPolypeptide Fifth Polypeptide Third Polypeptide First Polypeptide 4Fourth Polypeptide Fifth Polypeptide Third Polypeptide SecondPolypeptide 4 Fourth Polypeptide Fifth Polypeptide Third PolypeptideSixth Polypeptide 4 Fourth Polypeptide Fifth Polypeptide SixthPolypeptide First Polypeptide 4 Fourth Polypeptide Fifth PolypeptideSixth Polypeptide Second Polypeptide 4 Fourth Polypeptide FifthPolypeptide Sixth Polypeptide Third Polypeptide 4 Fourth PolypeptideSixth Polypeptide First Polypeptide Second Polypeptide 4 FourthPolypeptide Sixth Polypeptide First Polypeptide Third Polypeptide 4Fourth Polypeptide Sixth Polypeptide First Polypeptide Fifth Polypeptide4 Fourth Polypeptide Sixth Polypeptide Second Polypeptide FirstPolypeptide 4 Fourth Polypeptide Sixth Polypeptide Second PolypeptideThird Polypeptide 4 Fourth Polypeptide Sixth Polypeptide SecondPolypeptide Fifth Polypeptide 4 Fourth Polypeptide Sixth PolypeptideThird Polypeptide First Polypeptide 4 Fourth Polypeptide SixthPolypeptide Third Polypeptide Second Polypeptide 4 Fourth PolypeptideSixth Polypeptide Third Polypeptide Fifth Polypeptide 4 FourthPolypeptide Sixth Polypeptide Fifth Polypeptide First Polypeptide 4Fourth Polypeptide Sixth Polypeptide Fifth Polypeptide SecondPolypeptide 4 Fourth Polypeptide Sixth Polypeptide Fifth PolypeptideThird Polypeptide 4 Fifth Polypeptide First Polypeptide SecondPolypeptide Third Polypeptide 4 Fifth Polypeptide First PolypeptideSecond Polypeptide Fourth Polypeptide 4 Fifth Polypeptide FirstPolypeptide Second Polypeptide Sixth Polypeptide 4 Fifth PolypeptideFirst Polypeptide Third Polypeptide Second Polypeptide 4 FifthPolypeptide First Polypeptide Third Polypeptide Fourth Polypeptide 4Fifth Polypeptide First Polypeptide Third Polypeptide Sixth Polypeptide4 Fifth Polypeptide First Polypeptide Fourth Polypeptide SecondPolypeptide 4 Fifth Polypeptide First Polypeptide Fourth PolypeptideThird Polypeptide 4 Fifth Polypeptide First Polypeptide FourthPolypeptide Sixth Polypeptide 4 Fifth Polypeptide First PolypeptideSixth Polypeptide Second Polypeptide 4 Fifth Polypeptide FirstPolypeptide Sixth Polypeptide Third Polypeptide 4 Fifth PolypeptideFirst Polypeptide Sixth Polypeptide Fourth Polypeptide 4 FifthPolypeptide Second Polypeptide First Polypeptide Third Polypeptide 4Fifth Polypeptide Second Polypeptide First Polypeptide FourthPolypeptide 4 Fifth Polypeptide Second Polypeptide First PolypeptideSixth Polypeptide 4 Fifth Polypeptide Second Polypeptide ThirdPolypeptide First Polypeptide 4 Fifth Polypeptide Second PolypeptideThird Polypeptide Fourth Polypeptide 4 Fifth Polypeptide SecondPolypeptide Third Polypeptide Sixth Polypeptide 4 Fifth PolypeptideSecond Polypeptide Fourth Polypeptide First Polypeptide 4 FifthPolypeptide Second Polypeptide Fourth Polypeptide Third Polypeptide 4Fifth Polypeptide Second Polypeptide Fourth Polypeptide SixthPolypeptide 4 Fifth Polypeptide Second Polypeptide Sixth PolypeptideFirst Polypeptide 4 Fifth Polypeptide Second Polypeptide SixthPolypeptide Third Polypeptide 4 Fifth Polypeptide Second PolypeptideSixth Polypeptide Fourth Polypeptide 4 Fifth Polypeptide ThirdPolypeptide First Polypeptide Second Polypeptide 4 Fifth PolypeptideThird Polypeptide First Polypeptide Fourth Polypeptide 4 FifthPolypeptide Third Polypeptide First Polypeptide Sixth Polypeptide 4Fifth Polypeptide Third Polypeptide Second Polypeptide First Polypeptide4 Fifth Polypeptide Third Polypeptide Second Polypeptide FourthPolypeptide 4 Fifth Polypeptide Third Polypeptide Second PolypeptideSixth Polypeptide 4 Fifth Polypeptide Third Polypeptide FourthPolypeptide First Polypeptide 4 Fifth Polypeptide Third PolypeptideFourth Polypeptide Second Polypeptide 4 Fifth Polypeptide ThirdPolypeptide Fourth Polypeptide Sixth Polypeptide 4 Fifth PolypeptideThird Polypeptide Sixth Polypeptide First Polypeptide 4 FifthPolypeptide Third Polypeptide Sixth Polypeptide Second Polypeptide 4Fifth Polypeptide Third Polypeptide Sixth Polypeptide Fourth Polypeptide4 Fifth Polypeptide Fourth Polypeptide First Polypeptide SecondPolypeptide 4 Fifth Polypeptide Fourth Polypeptide First PolypeptideThird Polypeptide 4 Fifth Polypeptide Fourth Polypeptide FirstPolypeptide Sixth Polypeptide 4 Fifth Polypeptide Fourth PolypeptideSecond Polypeptide First Polypeptide 4 Fifth Polypeptide FourthPolypeptide Second Polypeptide Third Polypeptide 4 Fifth PolypeptideFourth Polypeptide Second Polypeptide Sixth Polypeptide 4 FifthPolypeptide Fourth Polypeptide Third Polypeptide First Polypeptide 4Fifth Polypeptide Fourth Polypeptide Third Polypeptide SecondPolypeptide 4 Fifth Polypeptide Fourth Polypeptide Third PolypeptideSixth Polypeptide 4 Fifth Polypeptide Fourth Polypeptide SixthPolypeptide First Polypeptide 4 Fifth Polypeptide Fourth PolypeptideSixth Polypeptide Second Polypeptide 4 Fifth Polypeptide FourthPolypeptide Sixth Polypeptide Third Polypeptide 4 Fifth PolypeptideSixth Polypeptide First Polypeptide Second Polypeptide 4 FifthPolypeptide Sixth Polypeptide First Polypeptide Third Polypeptide 4Fifth Polypeptide Sixth Polypeptide First Polypeptide Fourth Polypeptide4 Fifth Polypeptide Sixth Polypeptide Second Polypeptide FirstPolypeptide 4 Fifth Polypeptide Sixth Polypeptide Second PolypeptideThird Polypeptide 4 Fifth Polypeptide Sixth Polypeptide SecondPolypeptide Fourth Polypeptide 4 Fifth Polypeptide Sixth PolypeptideThird Polypeptide First Polypeptide 4 Fifth Polypeptide SixthPolypeptide Third Polypeptide Second Polypeptide 4 Fifth PolypeptideSixth Polypeptide Third Polypeptide Fourth Polypeptide 4 FifthPolypeptide Sixth Polypeptide Fourth Polypeptide First Polypeptide 4Fifth Polypeptide Sixth Polypeptide Fourth Polypeptide SecondPolypeptide 4 Fifth Polypeptide Sixth Polypeptide Fourth PolypeptideThird Polypeptide 4 Sixth Polypeptide First Polypeptide SecondPolypeptide Third Polypeptide 4 Sixth Polypeptide First PolypeptideSecond Polypeptide Fourth Polypeptide 4 Sixth Polypeptide FirstPolypeptide Second Polypeptide Fifth Polypeptide 4 Sixth PolypeptideFirst Polypeptide Third Polypeptide Second Polypeptide 4 SixthPolypeptide First Polypeptide Third Polypeptide Fourth Polypeptide 4Sixth Polypeptide First Polypeptide Third Polypeptide Fifth Polypeptide4 Sixth Polypeptide First Polypeptide Fourth Polypeptide SecondPolypeptide 4 Sixth Polypeptide First Polypeptide Fourth PolypeptideThird Polypeptide 4 Sixth Polypeptide First Polypeptide FourthPolypeptide Fifth Polypeptide 4 Sixth Polypeptide First PolypeptideFifth Polypeptide Second Polypeptide 4 Sixth Polypeptide FirstPolypeptide Fifth Polypeptide Third Polypeptide 4 Sixth PolypeptideFirst Polypeptide Fifth Polypeptide Fourth Polypeptide 4 SixthPolypeptide Second Polypeptide First Polypeptide Third Polypeptide 4Sixth Polypeptide Second Polypeptide First Polypeptide FourthPolypeptide 4 Sixth Polypeptide Second Polypeptide First PolypeptideFifth Polypeptide 4 Sixth Polypeptide Second Polypeptide ThirdPolypeptide First Polypeptide 4 Sixth Polypeptide Second PolypeptideThird Polypeptide Fourth Polypeptide 4 Sixth Polypeptide SecondPolypeptide Third Polypeptide Fifth Polypeptide 4 Sixth PolypeptideSecond Polypeptide Fourth Polypeptide First Polypeptide 4 SixthPolypeptide Second Polypeptide Fourth Polypeptide Third Polypeptide 4Sixth Polypeptide Second Polypeptide Fourth Polypeptide FifthPolypeptide 4 Sixth Polypeptide Second Polypeptide Fifth PolypeptideFirst Polypeptide 4 Sixth Polypeptide Second Polypeptide FifthPolypeptide Third Polypeptide 4 Sixth Polypeptide Second PolypeptideFifth Polypeptide Fourth Polypeptide 4 Sixth Polypeptide ThirdPolypeptide First Polypeptide Second Polypeptide 4 Sixth PolypeptideThird Polypeptide First Polypeptide Fourth Polypeptide 4 SixthPolypeptide Third Polypeptide First Polypeptide Fifth Polypeptide 4Sixth Polypeptide Third Polypeptide Second Polypeptide First Polypeptide4 Sixth Polypeptide Third Polypeptide Second Polypeptide FourthPolypeptide 4 Sixth Polypeptide Third Polypeptide Second PolypeptideFifth Polypeptide 4 Sixth Polypeptide Third Polypeptide FourthPolypeptide First Polypeptide 4 Sixth Polypeptide Third PolypeptideFourth Polypeptide Second Polypeptide 4 Sixth Polypeptide ThirdPolypeptide Fourth Polypeptide Fifth Polypeptide 4 Sixth PolypeptideThird Polypeptide Fifth Polypeptide First Polypeptide 4 SixthPolypeptide Third Polypeptide Fifth Polypeptide Second Polypeptide 4Sixth Polypeptide Third Polypeptide Fifth Polypeptide Fourth Polypeptide4 Sixth Polypeptide Fourth Polypeptide First Polypeptide SecondPolypeptide 4 Sixth Polypeptide Fourth Polypeptide First PolypeptideThird Polypeptide 4 Sixth Polypeptide Fourth Polypeptide FirstPolypeptide Fifth Polypeptide 4 Sixth Polypeptide Fourth PolypeptideSecond Polypeptide First Polypeptide 4 Sixth Polypeptide FourthPolypeptide Second Polypeptide Third Polypeptide 4 Sixth PolypeptideFourth Polypeptide Second Polypeptide Fifth Polypeptide 4 SixthPolypeptide Fourth Polypeptide Third Polypeptide First Polypeptide 4Sixth Polypeptide Fourth Polypeptide Third Polypeptide SecondPolypeptide 4 Sixth Polypeptide Fourth Polypeptide Third PolypeptideFifth Polypeptide 4 Sixth Polypeptide Fourth Polypeptide FifthPolypeptide First Polypeptide 4 Sixth Polypeptide Fourth PolypeptideFifth Polypeptide Second Polypeptide 4 Sixth Polypeptide FourthPolypeptide Fifth Polypeptide Third Polypeptide 4 Sixth PolypeptideFifth Polypeptide First Polypeptide Second Polypeptide 4 SixthPolypeptide Fifth Polypeptide First Polypeptide Third Polypeptide 4Sixth Polypeptide Fifth Polypeptide First Polypeptide Fourth Polypeptide4 Sixth Polypeptide Fifth Polypeptide Second Polypeptide FirstPolypeptide 4 Sixth Polypeptide Fifth Polypeptide Second PolypeptideThird Polypeptide 4 Sixth Polypeptide Fifth Polypeptide SecondPolypeptide Fourth Polypeptide 4 Sixth Polypeptide Fifth PolypeptideThird Polypeptide First Polypeptide 4 Sixth Polypeptide FifthPolypeptide Third Polypeptide Second Polypeptide 4 Sixth PolypeptideFifth Polypeptide Third Polypeptide Fourth Polypeptide 4 SixthPolypeptide Fifth Polypeptide Fourth Polypeptide First Polypeptide 4Sixth Polypeptide Fifth Polypeptide Fourth Polypeptide SecondPolypeptide 4 Sixth Polypeptide Fifth Polypeptide Fourth PolypeptideThird Polypeptide

Where n is 5, X moieties are selected from the following:

n X₁ X₂ X₃ X₄ X₅ 5 1st Polypeptide 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 5 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 5 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 5 1st Polypeptide 2nd Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 5 1st Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 5 1stPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 1st Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 3rd Polypeptide 5 1st Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 1st Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 5 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 1st Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 5 1st Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 5 1st Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 5 1stPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 1st Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 5 1st Polypeptide 2nd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 1st Polypeptide 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 5 1stPolypeptide 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 5 1st Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 3rd Polypeptide 5 1st Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 1st Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 5 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 5 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 5 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 5 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 5 1st Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 5 1stPolypeptide 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 5 1st Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 5 1st Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 2nd Polypeptide 5 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 1st Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 4th Polypeptide 5 1st Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 5 1st Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 5 1stPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 1st Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 5 1st Polypeptide 3rd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 1st Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 5 1stPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 5 1st Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5 1st Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 1st Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 5 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 1st Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 5 1st Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 5 1stPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 5 1st Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 5 1st Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 5 1stPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 5 1st Polypeptide 4th Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 5 1st Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5 1stPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 5 1st Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 1st Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 5 1st Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 1stPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 5 1st Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 5 1st Polypeptide 4th Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 5 1st Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 1stPolypeptide 4th Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 5 1st Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 1st Polypeptide 4th Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 5 1st Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 5 1stPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 5 1st Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5 1st Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 1st Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5 1stPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 5 1st Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 5 1st Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5 1stPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 1st Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5 1st Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 1st Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5 1stPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 5 1st Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 1st Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 5 1st Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 1stPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 1st Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5 1st Polypeptide 5th Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5 1st Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 1stPolypeptide 5th Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 5 1st Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 1st Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5 1st Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 2nd Polypeptide 5 1stPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 5 1st Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5 1st Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 5 1st Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5 1stPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 5 1st Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 5 1st Polypeptide 6th Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 5 1st Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5 1stPolypeptide 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 5 1st Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5 1st Polypeptide 6th Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 5 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 5 1stPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 5 1st Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 1st Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 5 1st Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 1stPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 5 1st Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 5 1st Polypeptide 6th Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 5 1st Polypeptide 6thPolypeptide 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 1stPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 5 1st Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 1st Polypeptide 6th Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 5 1st Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 5 1stPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 5 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 5 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 5 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 5 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 5 2nd Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 5 2ndPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 2nd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 2nd Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 5 2nd Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 5 2nd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 2nd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 1st Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 2nd Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 5 2ndPolypeptide 1st Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 5 2nd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 2nd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 5 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 5 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 5 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 1st Polypeptide 5 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 4th Polypeptide 5 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 5 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5 2nd Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 2nd Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 5th Polypeptide 5 2nd Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 2nd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 5 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 5 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 5 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 5 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 5 2nd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 5 2nd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 2nd Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 1st Polypeptide 5 2ndPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 5 2nd Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 2nd Polypeptide 4th Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 5 2nd Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5 2ndPolypeptide 4th Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 2nd Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5 2nd Polypeptide 4th Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 5 2nd Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 5 2nd Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5 2nd Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5 2nd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 5 2nd Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 5 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 5 2nd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5 2nd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5 2nd Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 2ndPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 2nd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5 2nd Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5 2ndPolypeptide 5th Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 5 2nd Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5 2nd Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5 2nd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 1st Polypeptide 5 2ndPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 5 2nd Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5 2nd Polypeptide 6th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 5 2nd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5 2ndPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 5 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 5 2nd Polypeptide 6th Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 5 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 5 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 5 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 1st Polypeptide 5 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 5 2nd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5 2nd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 5 2nd Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 2ndPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 5 2nd Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 2nd Polypeptide 6th Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 5 2nd Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 5 2ndPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 5 2nd Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 5 2nd Polypeptide 6th Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 5 2nd Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 2ndPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 5 3rd Polypeptide 1st Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 5 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 5 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 5 3rd Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 5 3rd Polypeptide 1stPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 5 3rdPolypeptide 1st Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 5 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 3rd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 3rd Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 4th Polypeptide 5 3rd Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 5 3rd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 3rd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 1st Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 3rd Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 5 3rdPolypeptide 1st Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 5 3rd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 3rd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 5 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 5 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 5 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 1st Polypeptide 5 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 4th Polypeptide 5 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 5 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 5 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 5 3rd Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 5th Polypeptide 5 3rd Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 3rd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 5 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 5 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 5 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 5 3rd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 5 3rd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 3rd Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 1st Polypeptide 5 3rdPolypeptide 4th Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 5 3rd Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 3rd Polypeptide 4th Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 5 3rd Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 3rdPolypeptide 4th Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 3rd Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 3rd Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 5 3rd Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 5 3rd Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5 3rd Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 3rd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 5 3rd Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 5 3rd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5 3rd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5 3rd Polypeptide 5thPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5 3rdPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 5 3rd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 5 3rd Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 3rdPolypeptide 5th Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 5 3rd Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 3rd Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 5 3rd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 1st Polypeptide 5 3rdPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 5 3rd Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5 3rd Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 5 3rd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 2nd Polypeptide 5 3rdPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 5 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 5 3rd Polypeptide 6th Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 5 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 5 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 5 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 1st Polypeptide 5 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 5 3rd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5 3rd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 5 3rd Polypeptide 6thPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5 3rdPolypeptide 6th Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 5 3rd Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 3rd Polypeptide 6th Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 5 3rd Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 2nd Polypeptide 5 3rdPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 5 3rd Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 5 3rd Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 5 3rd Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 3rdPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 5 4th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 5 4th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 5 4thPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 5 4th Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5 4th Polypeptide 1st Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 5 4th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 5 4thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 4th Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 5 4th Polypeptide 1st Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 5 4th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 5 4th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 4th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 5 4th Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 4thPolypeptide 1st Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 5 4th Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 5 4th Polypeptide 1st Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 5 4th Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 4thPolypeptide 1st Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 5 4th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 4th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 5 4th Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 5 4thPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 5 4th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 5th Polypeptide 5 4th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 3rd Polypeptide 5 4thPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 4th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 5 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 5 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 5 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 5 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 5 4th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 5 4th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 4th Polypeptide 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 1st Polypeptide 5 4thPolypeptide 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 5 4th Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 4th Polypeptide 2nd Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 5 4th Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5 4thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 4th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5 4th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 5 4th Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 4thPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 5 4th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 5th Polypeptide 5 4th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 4th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 2nd Polypeptide 5 4thPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 5 4th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 5 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 5 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 5 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 5 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 5 4th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 5 4th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 5 4th Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 1st Polypeptide 5 4thPolypeptide 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 5 4th Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 5 4th Polypeptide 3rd Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 5 4th Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 4thPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 5 4th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 4th Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 5 4th Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 5 4thPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 5 4th Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 4th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 4th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 4thPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 5 4th Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 4th Polypeptide 5th Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 5 4th Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 4thPolypeptide 5th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 4th Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 4th Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 4th Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 4thPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 5 4th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 4th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 5 4th Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 4thPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 4th Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 5 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 5 4th Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 4thPolypeptide 5th Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 5 4th Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 4th Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 4th Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 1st Polypeptide 5 4thPolypeptide 5th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 5 4th Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 4th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 5 4th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 4thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 5 4th Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 5 4th Polypeptide 6th Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 5 4th Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 4thPolypeptide 6th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 5 4th Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 4th Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 5 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 1st Polypeptide 5 4thPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 5 4th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 4th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 5 4th Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 4thPolypeptide 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 5 4th Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 5 4th Polypeptide 6th Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 5 4th Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 2nd Polypeptide 5 4thPolypeptide 6th Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 5 4th Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 5 4th Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 4th Polypeptide 6thPolypeptide 5th Polypeptide 3rd Polypeptide 1st Polypeptide 5 4thPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 5 5th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5 5th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 5th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5 5thPolypeptide 1st Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 5 5th Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5 5th Polypeptide 1st Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 5 5th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5 5thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 5th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5 5th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 5th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 5 5th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 5th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 5 5th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 5thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 5th Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5 5th Polypeptide 1st Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5 5th Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 5thPolypeptide 1st Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 5 5th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 5th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5 5th Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 2nd Polypeptide 5 5thPolypeptide 1st Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 5 5th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5 5th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5 5th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5 5thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 5 5th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 5 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 5 5th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5 5th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5 5th Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 5thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 5 5th Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5 5th Polypeptide 2nd Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5 5th Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5 5thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 5 5th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5 5th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5 5th Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 1st Polypeptide 5 5thPolypeptide 2nd Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 5 5th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5 5th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 5th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 2nd Polypeptide 5 5thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 5 5th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 5 5th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5 5th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5 5th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5 5thPolypeptide 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 5 5th Polypeptide 3rd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5 5th Polypeptide 3rd Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 5 5th Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 5thPolypeptide 3rd Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 5 5th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 5th Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 5 5th Polypeptide 3rdPolypeptide 6th Polypeptide 4th Polypeptide 1st Polypeptide 5 5thPolypeptide 3rd Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 5 5th Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 5th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5 5th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 5thPolypeptide 4th Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 5 5th Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5 5th Polypeptide 4th Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 5 5th Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 5thPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 5 5th Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 5th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5 5th Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5 5thPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 5 5th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 5th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 5 5th Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 5thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 5 5th Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 5 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 5 5th Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5 5thPolypeptide 4th Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 5 5th Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5 5th Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 5th Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 1st Polypeptide 5 5thPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 5 5th Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 5th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 5 5th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 5thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 5 5th Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5 5th Polypeptide 6th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 5 5th Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 5thPolypeptide 6th Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 5 5th Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 5th Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 5 5th Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 1st Polypeptide 5 5thPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 5 5th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 5th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 5 5th Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 5thPolypeptide 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 5 5th Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 5th Polypeptide 6th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 5 5th Polypeptide 6thPolypeptide 4th Polypeptide 1st Polypeptide 2nd Polypeptide 5 5thPolypeptide 6th Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 5 5th Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5 5th Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 5th Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 5thPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 5 6th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5 6th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 5 6th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5 6thPolypeptide 1st Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 5 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 5 6th Polypeptide 1st Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 5 6th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5 6thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 5 6th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5 6th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 5 6th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 5 6thPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 5 6th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5 6th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 5 6th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5 6thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 5 6th Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 5 6th Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 5 6th Polypeptide 1stPolypeptide 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 5 6thPolypeptide 1st Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 5 6th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 5 6th Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 5 6th Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 5 6thPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 5 6th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5 6th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 5 6th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5 6thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 5 6th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 5 6th Polypeptide 2nd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 5 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 5 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 5 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 1st Polypeptide 5 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 5 6th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5 6th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 5 6th Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 6thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 5 6th Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 6th Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 5 6th Polypeptide 2ndPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 5 6thPolypeptide 2nd Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 5 6th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 5 6th Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 5 6th Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 6thPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 5 6th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5 6th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 5 6th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 2nd Polypeptide 5 6thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 5 6th Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 5 6th Polypeptide 3rd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 5 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 5 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 5 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 1st Polypeptide 5 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 5 6th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5 6th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 5 6th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5 6thPolypeptide 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 5 6th Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 5 6th Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 5 6th Polypeptide 3rdPolypeptide 5th Polypeptide 1st Polypeptide 2nd Polypeptide 5 6thPolypeptide 3rd Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 5 6th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 5 6th Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 5 6th Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 5 6thPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 5 6th Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 6th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 5 6th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 6thPolypeptide 4th Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 5 6th Polypeptide 4th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 5 6th Polypeptide 4th Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 5 6th Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 6thPolypeptide 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 5 6th Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 6th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 5 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 1st Polypeptide 5 6thPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 5 6th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 6th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 5 6th Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 6thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 5 6th Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 5 6th Polypeptide 4th Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 5 6th Polypeptide 4thPolypeptide 5th Polypeptide 1st Polypeptide 2nd Polypeptide 5 6thPolypeptide 4th Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 5 6th Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 5 6th Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 6th Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 1st Polypeptide 5 6thPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 5 6th Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5 6th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 5 6th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5 6thPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 5 6th Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5 6th Polypeptide 5th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 5 6th Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5 6thPolypeptide 5th Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 5 6th Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5 6th Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 5 6th Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 1st Polypeptide 5 6thPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 5 6th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5 6th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 5 6th Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5 6thPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 5 6th Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5 6th Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 5 6th Polypeptide 5thPolypeptide 4th Polypeptide 1st Polypeptide 2nd Polypeptide 5 6thPolypeptide 5th Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 5 6th Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5 6th Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 5 6th Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5 6thPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide

Where n is 6, X moieties are selected from the following:

n X₁ X₂ X₃ X₄ X₅ X₆ 6 1st Polypeptide 2nd Polypeptide 3rd Polypeptide4th Polypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 2nd Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 3rd Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 3rdPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 1st Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 1st Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 1st Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 1st Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 1st Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 1st Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 1st Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 1st Polypeptide 6thPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 1st Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 1st Polypeptide 6thPolypeptide 5th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 1st Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 1st Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 4th Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 2nd Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 2nd Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 2nd Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 2nd Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 2nd Polypeptide 6thPolypeptide 5th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 2nd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 2nd Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 3rd Polypeptide 1st Polypeptide 2nd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 1st Polypeptide 2nd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 4th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 4thPolypeptide 5th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 4th Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 4thPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 3rd Polypeptide 5thPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 5thPolypeptide 4th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 5th Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 5thPolypeptide 6th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 3rd Polypeptide 6thPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 6thPolypeptide 4th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 3rd Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 3rd Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 3rd Polypeptide 6thPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 3rd Polypeptide 6th Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 3rd Polypeptide 6thPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 1st Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 1st Polypeptide 5th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 1stPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 2nd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 2ndPolypeptide 6th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 5th Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 5thPolypeptide 6 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 5th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 5th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 3rdPolypeptide 5th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 3rd Polypeptide 6th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 3rdPolypeptide 6th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 5th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 5thPolypeptide 1st Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 4th Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 5thPolypeptide 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 4th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 4th Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 5thPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 5thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 5th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 5thPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 5th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 5thPolypeptide 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 6th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 6thPolypeptide 1st Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 4th Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 6thPolypeptide 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 4th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 4th Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 6thPolypeptide 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 4th Polypeptide 6thPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 4th Polypeptide 6th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 4th Polypeptide 6thPolypeptide 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 4th Polypeptide 6th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 4th Polypeptide 6thPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 1st Polypeptide 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 1st Polypeptide 2nd Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 1st Polypeptide 3rd Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 1st Polypeptide 4th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 1stPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 1stPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 1stPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 2nd Polypeptide 1st Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 2nd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 2ndPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 2ndPolypeptide 6th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 2ndPolypeptide 6th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 3rd Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 3rd Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 4th Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 4thPolypeptide 6 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 4th Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 4th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 3rdPolypeptide 4th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 3rdPolypeptide 6th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 3rdPolypeptide 6th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 3rd Polypeptide 6th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 3rdPolypeptide 6th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 4th Polypeptide 1st Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 4thPolypeptide 1st Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 6th Polypeptide 3rdPolypeptide 6 5th Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 2nd Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 4thPolypeptide 2nd Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 3rd Polypeptide 1st Polypeptide 6th Polypeptide 2ndPolypeptide 6 5th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 6th Polypeptide 6 5th Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 6th Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 3rd Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 4thPolypeptide 3rd Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 6th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 4thPolypeptide 6th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 4th Polypeptide 6th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 4thPolypeptide 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 4th Polypeptide 6th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 4thPolypeptide 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 1st Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 6th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 1st Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 6th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 6thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 6th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 5th Polypeptide 6th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 6thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 5th Polypeptide 6th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 5th Polypeptide 6thPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 6thPolypeptide 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 5th Polypeptide 6thPolypeptide 4th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 5th Polypeptide 6th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 5th Polypeptide 6thPolypeptide 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 5th Polypeptide 6th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 5th Polypeptide 6thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 1st Polypeptide 2nd Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 1st Polypeptide 2nd Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 1st Polypeptide 2nd Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 1st Polypeptide 3rd Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 1st Polypeptide 3rd Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 1stPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 1st Polypeptide 4th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 1stPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 1stPolypeptide 5th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 1stPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 2nd Polypeptide 1st Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 2nd Polypeptide 1st Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 2nd Polypeptide 1st Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 2nd Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 2ndPolypeptide 4th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 2nd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 2ndPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 2ndPolypeptide 5th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 2ndPolypeptide 5th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 1st Polypeptide 2ndPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 3rd Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 3rd Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 4th Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 4thPolypeptide 6 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 4th Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 3rdPolypeptide 4th Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 4th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 3rdPolypeptide 4th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 3rdPolypeptide 5th Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 3rdPolypeptide 5th Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 3rd Polypeptide 5th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 3rdPolypeptide 5th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 4th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 4th Polypeptide 1st Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 4thPolypeptide 1st Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 2nd Polypeptide 1st Polypeptide 5th Polypeptide 3rdPolypeptide 6 6th Polypeptide 4th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 2nd Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 4thPolypeptide 2nd Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 3rd Polypeptide 1st Polypeptide 5th Polypeptide 2ndPolypeptide 6 6th Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 5th Polypeptide 6 6th Polypeptide 4thPolypeptide 3rd Polypeptide 2nd Polypeptide 5th Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 3rd Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 4thPolypeptide 3rd Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 5th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 4thPolypeptide 5th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 4th Polypeptide 5th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 4thPolypeptide 5th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 4th Polypeptide 5th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 4thPolypeptide 5th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 1st Polypeptide 2ndPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 1st Polypeptide 2nd Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 5th Polypeptide 1st Polypeptide 3rdPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 1st Polypeptide 3rd Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 5th Polypeptide 1st Polypeptide 4thPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 5thPolypeptide 1st Polypeptide 4th Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 5th Polypeptide 2nd Polypeptide 1stPolypeptide 3rd Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 2nd Polypeptide 1st Polypeptide 4th Polypeptide 3rdPolypeptide 6 6th Polypeptide 5th Polypeptide 2nd Polypeptide 3rdPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 2nd Polypeptide 3rd Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 2nd Polypeptide 4thPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 5thPolypeptide 2nd Polypeptide 4th Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 3rd Polypeptide 1stPolypeptide 2nd Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 3rd Polypeptide 1st Polypeptide 4th Polypeptide 2ndPolypeptide 6 6th Polypeptide 5th Polypeptide 3rd Polypeptide 2ndPolypeptide 1st Polypeptide 4th Polypeptide 6 6th Polypeptide 5thPolypeptide 3rd Polypeptide 2nd Polypeptide 4th Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 3rd Polypeptide 4thPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 5thPolypeptide 3rd Polypeptide 4th Polypeptide 2nd Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 4th Polypeptide 1stPolypeptide 2nd Polypeptide 3rd Polypeptide 6 6th Polypeptide 5thPolypeptide 4th Polypeptide 1st Polypeptide 3rd Polypeptide 2ndPolypeptide 6 6th Polypeptide 5th Polypeptide 4th Polypeptide 2ndPolypeptide 1st Polypeptide 3rd Polypeptide 6 6th Polypeptide 5thPolypeptide 4th Polypeptide 2nd Polypeptide 3rd Polypeptide 1stPolypeptide 6 6th Polypeptide 5th Polypeptide 4th Polypeptide 3rdPolypeptide 1st Polypeptide 2nd Polypeptide 6 6th Polypeptide 5thPolypeptide 4th Polypeptide 3rd Polypeptide 2nd Polypeptide 1stPolypeptide

Where n is 7, any combination of the first, second, third, fourth,fifth, sixth and seventh polypeptide may be included in any order, asdiscussed above when n is 2, 3, 4, 5, or 6.

The invention also provides a cell (typically a bacterium) whichexpresses at least two of:

-   -   a) a first polypeptide comprising a first amino acid sequence,        wherein the first amino acid sequence comprises an amino acid        sequence (i) having at least a% sequence identity to SEQ ID NO:1        and/or (ii) consisting of a fragment of at least t contiguous        amino acids from SEQ ID NO:1 or from a sequence having at least        a% sequence identity to SEQ ID NO:1;    -   b) a second polypeptide comprising a second amino acid sequence,        wherein the second amino acid sequence comprises an amino acid        sequence (i) having at least b% sequence identity to SEQ ID NO:2        and/or (ii) consisting of a fragment of at least u contiguous        amino acids from SEQ ID NO:2 or from a sequence having at least        b% sequence identity to SEQ ID NO:1;    -   c) a third polypeptide comprising a third amino acid sequence,        wherein the third amino acid sequence comprises an amino acid        sequence (i) having at least c% sequence identity to SEQ ID NO:3        and/or (ii) consisting of a fragment of at least v contiguous        amino acids from SEQ ID NO:3 or from a sequence having at least        c% sequence identity to SEQ ID NO:3;    -   d) a fourth polypeptide comprising a fourth amino acid sequence,        wherein the fourth amino acid sequence comprises an amino acid        sequence (i) having at least d% sequence identity to SEQ ID NO:4        and/or (ii) consisting of a fragment of at least w contiguous        amino acids from SEQ ID NO:4 or from a sequence having at least        d% sequence identity to SEQ ID NO:4;    -   e) a fifth polypeptide comprising a fifth amino acid sequence,        wherein the fifth amino acid sequence comprises an amino acid        sequence (i) having at least e% sequence identity to SEQ ID NO:5        and/or (ii) consisting of a fragment of at least x contiguous        amino acids from SEQ ID NO:5 or from a sequence having at least        e% sequence identity to SEQ ID NO:5; and/or    -   f) a sixth polypeptide comprising a sixth amino acid sequence,        wherein the sixth amino acid sequence comprises an amino acid        sequence (i) having at least f% sequence identity to SEQ ID NO:6        and/or (ii) consisting of a fragment of at least y contiguous        amino acids from SEQ ID NO:6 or from a sequence having at least        f% sequence identity to SEQ ID NO:6; and/or    -   g) a seventh polypeptide comprising a sixth amino acid sequence,        wherein the sixth amino acid sequence comprises an amino acid        sequence (i) having at least g% sequence identity to SEQ ID NO:7        and/or (ii) consisting of a fragment of at least z contiguous        amino acids from SEQ ID NO:6 or from a sequence having at least        g% sequence identity to SEQ ID NO:7

The cell may express 2, 3, 4, 5, 6 or all 7 of the seven amino acidsequences.

The First, Second, Third, Fourth, Fifth, Sixth and Seventh Amino AcidSequences

The value of a is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98,99 or more. The value of b is at least 75 e.g. 80, 85, 90, 92, 94, 95,96, 97, 98, 99 or more. The value of c is at least 75 e.g. 80, 85, 90,92, 94, 95, 96, 97, 98, 99 or more. The value of d is at least 75 e.g.80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of e is atleast 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more. The valueoff is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more.The value of g is at least 75 e.g. 80, 85, 90, 92, 94, 95, 96, 97, 98,99 or more. The values of a, b, c, d, e, f and g may be the same ordifferent. In some embodiments, a b, c, d, e and f are identical.Typically, a, b, c, d, e, f and g are at least 90 e.g. at least 95.

The value oft is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250. The value of u is atleast 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250. The value of v is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250. The value of w is at least 7 e.g. 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250. Thevalue of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 225, 250. The value of y is atleast 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250. The value of z is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250. The values of t, u, v, w, x, y and z may be the same ordifferent. In some embodiments, t, u, v, w, x, y and z are identical.

Fragments preferably comprise an epitope from the respective SEQ ID NO:sequence. Other useful fragments lack one or more amino acids (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of the respective SEQ ID NO: whileretaining at least one epitope thereof. Truncation by 20-25 amino acidsat the N-terminus is convenient e.g. removal of amino acids 1-29 of anyof SEQ ID NOs: 1 to 7 which constitute a leader peptide and/or removalof the C-terminal 35 amino acids of any of SEQ ID NOS:1-7 whichconstitute an LPXTG (SEQ ID NO:272) anchor.

The GBS59 protein can be split into four domains (D1 to D4) between theend of its leader peptide and the start of its LPXTG (SEQ ID NO:272)anchor. These four domains are as follows in SEQ ID NOs: 1 to 7, and thepositions in further GBS59 sequences which correspond to these residuescan readily be identified by alignment:

D1 D2 D3 D4 SEQ ID NO: 1 30-175 169-369 363-509 503-670 (2603) (SEQ IDNO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) SEQ ID NO: 230-162 156-338 332-499 472-640 (515) (SEQ ID NO: 12) (SEQ ID NO: 13)(SEQ ID NO: 14) (SEQ ID NO: 15) SEQ ID NO: 3 30-162 155-337 331-474468-639 (cjb111) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ IDNO: 19) SEQ ID NO: 4 30-158 152-350 343-493 487-658 (h36b) (SEQ ID NO:20) (SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID NO: 23) SEQ ID NO: 5 30-172166-365 359-507 501-669 (CJB110) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ IDNO: 26) (SEQ ID NO: 27) SEQ ID NO: 6 30-168 162-344 338-480 475-647(DK21) (SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID NO: 31)SEQ ID NO: 7 30-162 155-337 331-474 468-639 (NEM316) (SEQ ID NO: 32)(SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35)

Based on protection studies, useful fragments of GBS59 may retainepitopes from at least domain D3. The first, second, third, fourth,fifth, sixth or seventh amino acid sequences used in the immunogeniccompositions and polypeptides of the invention may therefore consist offragments of SEQ ID NOS: 1, 2, 3, 4, 5, 6 or 7 comprising domain D3, asidentified above.

Fragments of GBS59 may retain domains D4, D2 and/or D1 in addition toepitopes from domain D3. The first, second, third, fourth, fifth, sixthor seventh amino acid sequences used in the immunogenic compositions andpolypeptides of the invention may therefore consist of fragments of SEQID NOS: 1, 2, 3, 4, 5, 6 or 7 comprising i) domains D2 and D3; ii)domains D3 and D4; iii) domains D1, D2 and D3; or iv) domains D2, D3 andD4, as identified above.

Sub-fragments of these domains that retain epitopes required forimmunogenicity may be used instead of the complete domains, The first,second, third, fourth, fifth, sixth or seventh amino acid sequences usedin the immunogenic compositions and polypeptides of the invention maythus consist of fragments of SEQ ID NOS: 1, 2, 3, 4, 5, 6 or 7comprising sub-fragments of domain D3 (and optionally of domains D1, D3,and/or D4) that retain epitopes required for immunogenicity. Examples ofsub-fragments of domains D3 and D4 that may be present in the first,second, third, fourth, fifth, sixth or seventh amino acid sequences usedin the immunogenic compositions and polypeptides of the invention areidentified below. The sub-fragments of domain D3 identified below (SEQID NOs: 36, 38, 40, 42, 44, 46 and 48) are surface-exposed fragments.Smaller epitopes within these surface-exposed fragments can be readilyidentified by the skilled person and used in the compositions of theinvention. For example, two monoclonal antibodies (17C4/A3 and 4H11/B7,SEQ ID NOs: 262-269) have been found to bind an epitope comprising aminoacids 411-436 (SEQ ID NO: 270) within the D3 sub-fragment from the 515clade (SEQ ID NO: 38, fragment of SEQ ID NO: 2). The sub-fragments ofdomain D4 identified below comprise the two helices (referred to hereinas D4H) present at the N-terminal of domain D4 and not the remainder ofthe D4 domain. These helices are predicted to be surface-exposed.

D3 sub-fragments D4 sub-fragments (helices) SEQ ID NO: 1 363-483 484-588(2603) (SEQ ID NO: 36) (SEQ ID NO: 37) SEQ ID NO: 2 332-447 448-554(515) (SEQ ID NO: 38) (SEQ ID NO: 39) 411-436 (SEQ ID NO: 270) SEQ IDNO: 3 331-446 447-553 (cjb111) (SEQ ID NO: 40) (SEQ ID NO: 41) SEQ IDNO: 4 343-465 466-572 (h36b) (SEQ ID NO: 42) (SEQ ID NO: 43) SEQ ID NO:5 359-481 482-588 (CJB110) (SEQ ID NO: 44) (SEQ ID NO: 45) SEQ ID NO: 6338-453 454-561 (DK21) (SEQ ID NO: 46) (SEQ ID NO: 47) SEQ ID NO: 7331-446 447-553 (NEM316) (SEQ ID NO: 48) (SEQ ID NO: 49)

Suitable fragments of SEQ ID NO:1 are SEQ ID NOS:50-53.

Suitable fragments of SEQ ID NO:2 are SEQ ID NOS: 54-57.

Suitable fragments of SEQ ID NO:3 are SEQ ID NOS: 58-61.

Suitable fragments of SEQ ID NO:4 are SEQ ID NOS:62-65.

Suitable fragments of SEQ ID NO:5 are SEQ ID NOS:66-69.

Suitable fragments of SEQ ID NO:6 are SEQ ID NOS:70-73.

Suitable fragments of SEQ ID NO:7 are SEQ ID NOS: 74-77.

These fragments contain combinations of domains D2, D3, and D4 (or D4H)as set out below:

D3 + D4 D3 + D4H D2 + D3 + D4 D2 + D3 + D4H SEQ ID NO: 1 SEQ ID NO: 50SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 (2603) SEQ ID NO: 2 SEQ ID NO:54 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 57 (515) SEQ ID NO: 3 SEQ IDNO: 58 SEQ ID NO: 59 SEQ ID NO: 60 SEQ ID NO: 61 (cjb111) SEQ ID NO: 4SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 SEQ ID NO: 65 (h36b) SEQ IDNO: 5 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68 SEQ ID NO: 69 (CJB110)SEQ ID NO: 6 SEQ ID NO: 70 SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 73(DK21) SEQ ID NO: 7 SEQ ID NO: 74 SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO:77 (NEWM316)

In some cases, even smaller fragments may be used. For example, thethird amino acid sequence of the invention may consist of a fragment ofSEQ ID NO:3 comprising SEQ ID NO:78 (amino acids 411 to 436 of SEQ IDNO:3).

Where fragments are used, the fragment of at least t contiguous aminoacids from SEQ ID NO: 1 should not also be present within SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO:4, SEQ NO:5, SEQ ID NO:6 nor within SEQ ID NO:7.Similarly, the fragment of at least u contiguous amino acids from SEQ IDNO: 2 should not also be present within SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 nor within SEQ ID NO:7. Similarly, thefragment of at least v contiguous amino acids from SEQ ID NO: 3 shouldnot also be present within SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 nor within SEQ ID NO: 7. Similarly, the fragment ofat least w contiguous amino acids from SEQ ID NO: 4 should not also bepresent within SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:6 nor within SEQ ID NO: 7. Similarly, the fragment of at least xcontiguous amino acids from SEQ ID NO: 5 should not also be presentwithin SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6nor within SEQ ID NO: 7. Similarly, the fragment of at least ycontiguous amino acids from SEQ ID NO: 6 should not also be presentwithin SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5nor within SEQ ID NO: 7. Similarly, the fragment of at least zcontiguous amino acids from SEQ ID NO: 7 should not also be presentwithin SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5nor within SEQ ID NO: 6.In some embodiments, when a fragment from one ofSEQ ID NOs: 1 to 7 is aligned as a contiguous sequence against the othersix SEQ ID NOs, the identity between the fragment and each of the othersix SEQ ID NOs is less than 75% e.g. less than 60%, less than 50%, lessthan 40%, less than 30%.

A polypeptide comprising the first amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1 (strain 2603). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 3, the wild-type GBS protein having amino acidsequence SEQ ID NO: 4, the wild-type GBS protein having amino acidsequence SEQ ID NO: 5, the wild-type GBS protein having amino acidsequence SEQ ID NO: 6, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 7.

A polypeptide comprising the second amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 2 (strain 515). In some embodiments these antibodiesdo not bind to the wild-type GBS protein having amino acid sequence SEQID NO: 1, the wild-type GBS protein having amino acid sequence SEQ IDNO: 3, the wild-type GBS protein having amino acid sequence SEQ ID NO:4, the wild-type GBS protein having amino acid sequence SEQ ID NO: 5,the wild-type GBS protein having amino acid sequence SEQ ID NO: 6, orthe wild-type GBS protein having amino acid sequence SEQ ID NO: 7.

A polypeptide comprising the third amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 3 (strain cjb111). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1, the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 4, the wild-type GBS protein having amino acidsequence SEQ ID NO: 5, the wild-type GBS protein having amino acidsequence SEQ ID NO: 6, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 7.

A polypeptide comprising the fourth amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 4 (strain h36b). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1, the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 3, the wild-type GBS protein having amino acidsequence SEQ ID NO: 5, the wild-type GBS protein having amino acidsequence SEQ ID NO: 6, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 7.

A polypeptide comprising the fifth amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 5 (strain CJB110). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1, the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 3, the wild-type GBS protein having amino acidsequence SEQ ID NO: 4, the wild-type GBS protein having amino acidsequence SEQ ID NO: 6, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 7.

A polypeptide comprising the sixth amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 6 (strain DK21). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1, the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 3, the wild-type GBS protein having amino acidsequence SEQ ID NO: 4, the wild-type GBS protein having amino acidsequence SEQ ID NO: 5, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 7.

A polypeptide comprising the seventh amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 7 (strain NEM316). In some embodiments theseantibodies do not bind to the wild-type GBS protein having amino acidsequence SEQ ID NO: 1, the wild-type GBS protein having amino acidsequence SEQ ID NO: 2, the wild-type GBS protein having amino acidsequence SEQ ID NO: 3, the wild-type GBS protein having amino acidsequence SEQ ID NO: 4, the wild-type GBS protein having amino acidsequence SEQ ID NO: 5, or the wild-type GBS protein having amino acidsequence SEQ ID NO: 6.

Although the first, second, third, fourth, fifth, sixth and seventhamino acid sequences may share some sequences in common, overall theyhave different amino acid sequences.

Amino acid sequences used with the invention, may, compared to SEQ IDNOs: 1, 2, 3, 4, 5, 6, or 7 or fragments thereof include one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acidreplacements i.e. replacements of one amino acid with another which hasa related side chain. Genetically-encoded amino acids are generallydivided into four families: (1) acidic i.e. aspartate, glutamate; (2)basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Ingeneral, substitution of single amino acids within these families doesnot have a major effect on the biological activity. The polypeptides mayhave one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single aminoacid deletions relative to a reference sequence. The polypeptides mayalso include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to areference sequence.

In particular, the amino acid sequences of the invention may comprisesubstitutions amino acid residues involved in isopeptide bond formationwithin GBS59, identified in the table below.

Isopeptide Isopeptide Isopeptide bond amino bond amino bond amino acidsin D2 acids in D3 acids in D4 2603 K212, N356 K386, N473 K499, N666 (SEQID NO: 1) 515 K199, N325 K355, N437 K463, N636 (SEQ ID NO: 2) CJB111K198, N324 K354, N436 K462, N635 (SEQ ID NO: 3) H36B K195, N336 K366,N455 K481, N654 (SEQ ID NO: 4) CJB110 K209, N352 K382, N471 K497, N665(SEQ ID NO: 5) DK21 K205, N331 K361, N443 K469, N643 (SEQ ID NO: 6)NEM316 K198, N324 K354, N436 K462, N635 (SEQ ID NO: 7)

The data presented in the examples demonstrate that mutation of theseresidues to disrupt isopeptide bond formation does not adversely affectthe immunogenicity of the polypeptide. Accordingly, the polypeptides maycomprise substitutions at one or more the lysine residues or asparagineresidues recited in the table above. In some embodiments, the lysineresidues may be substituted by alanine residues.

A polypeptide used with the invention may comprise an amino acidsequence that:

-   -   (a) is identical (i.e. 100% identical) to SEQ ID NO: 1, 2, 3, 4,        5, 6 or 7, or is identical to a SEQ ID NO: 1, 2, 3, 4, 5, 6 or        7;    -   (b) shares sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6 or        7, or shares identity with a fragment of SEQ ID NO:1, 2, 3, 4,        5, 6 or 7;    -   (c) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single amino        acid alterations (deletions, insertions, substitutions), which        may be at separate locations or may be contiguous, as compared        to the sequences of (a) or (b); and    -   (d) when aligned SEQ ID 1, 2, 3, 4, 5, 6 or 7 using a pairwise        alignment algorithm, each moving window of x amino acids from        N-terminus to C-terminus (such that for an alignment that        extends top amino acids, where p>x, there are p−x+1 such        windows) has at least x·y identical aligned amino acids, where:        x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,        100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80,        0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98,        0.99; and if x·y is not an integer then it is rounded up to the        nearest integer. The preferred pairwise alignment algorithm is        the Needleman-Wunsch global alignment algorithm [3], using        default parameters (e.g. with Gap opening penalty=10.0, and with        Gap extension penalty=0.5, using the EBLOSUM62 scoring matrix).        This algorithm is conveniently implemented in the needle tool in        the EMBOSS package [4].

Within group (c), deletions or substitutions may be at the N-terminusand/or C-terminus, or may be between the two termini. Thus a truncationis an example of a deletion. Truncations may involve deletion of up to40 (or more) amino acids at the N-terminus and/or C-terminus.

Hybrid Polypeptides

Different GBS59 clades used in the invention do not have to be presentas separate polypeptides but can instead be expressed as a singlepolypeptide chain (a ‘hybrid’ polypeptide or ‘chimera’). Hybridpolypeptides offer two main advantages: first, a polypeptide that may beunstable or poorly expressed on its own can be assisted by adding asuitable hybrid partner that overcomes the problem; second, commercialmanufacture is simplified as only one expression and purification needto be employed in order to produce two polypeptides which are bothantigenically useful.

Hybrid polypeptides can include sequences from only GBS59 antigens butin other embodiments can include non-GBS59 antigens (usually non-GBS59antigens from GBS), such as other pilus subunits. If non-GBS59 antigensare present these may be to the N-terminus of any 2, 3, 4, 5, 6 or 7GBS59 sequences, to the C-terminus of any 2, 3, 4, 5, 6 or 7 GBS59sequences, or may be between two GBS59 sequences in a hybrid polypeptidecontaining 2, 3, 4, 5, 6 or 7 GBS59 sequences.

Different hybrid polypeptides may be mixed together in a singleformulation. Hybrids may be combined with non-hybrid GBS59 antigens orother non-GBS59 antigens.

Hybrid polypeptides may be represented by the formulaNH₂-A-{—X-L-}_(n)-B—COOH.

If a —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the—X— moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of {—X-L-} linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise shortpeptide sequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more,e.g. SEQ ID NO:79). Other suitable linker amino acid sequences will beapparent to those skilled in the art. Useful linkers are GSGS (SEQ IDNO:80), GSGGGG (SEQ ID NO: 81) or GSGSGGGG (SEQ ID NO: 82), with theGly-Ser dipeptide being formed from a BamHI restriction site, thusaiding cloning and manipulation, and the (Gly)₄ tetrapeptide being atypical poly-glycine linker. Other suitable linkers, particularly foruse as the final L_(n) are a Leu-Glu dipeptide or Gly-Ser. Linkers willusually contain at least one glycine residue to facilitate structuralflexibility e.g. a -L- moiety may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more glycine residues. Such glycines may be arranged to include atleast two consecutive glycines in a Gly-Gly dipeptide sequence, or alonger oligo-Gly sequence i.e. Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9,10 or more.

-A—is an optional N-terminal amino acid sequence. This will typically beshort (e.g. 40 or fewer amino acids i.e. 40, 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 20 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includeleader sequences to direct protein trafficking, or short peptidesequences which facilitate cloning or purification (e.g. histidine tagsi.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine e.g. Met-Ala-Ser, or a single Metresidue. In a nascent polypeptide the -A—moiety can provide thepolypeptide's N-terminal methionine (formyl-methionine, fMet, inbacteria). One or more amino acids may be cleaved from the N-terminus ofa nascent -A- moiety, however, such that the -A- moiety in a maturepolypeptide of the invention does not necessarily include a N-terminalmethionine.

—B— is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more, such as SEQ ID NO: 79),or sequences which enhance protein stability. Other suitable C-terminalamino acid sequences will be apparent to those skilled in the art, suchas a glutathione-S-transferase, thioredoxin, 14 kDa fragment of S.aureus protein A, a biotinylated peptide, a maltose-binding protein, anenterokinase flag, etc.

It is preferred that -A-, —B— and -L- sequences do not include asequence that shares 10 or more contiguous amino acids in common with ahuman polypeptide sequence.

In some embodiments, a -L- moiety comprises a non-GBS59 antigen. In someembodiments, the -A- moiety comprises a non-GBS59 antigen, and in somethe —B— moiety comprises a non-GBS59 antigen.

The invention also provides nucleic acid which encodes a hybridpolypeptide of the invention.

Of the various X and L moieties, useful combinations include, but arenot limited to:

SEQ ID X1* L1* X2* L2* X3* L3 X4 L4 X5 L5 X6 83 38 80 42 80 40 80 36 8044 80 46 (Fusion D3 flag 515 D3 flag D3 flag D3 flag D3 D3 E) h36bcjb111 2603 fragCJB110 fragDK21 84 55 80 59 80 67 80 51 80 63 80 71(Fusion D3 + D4H D3 + D4H D3 + D4H D3 + D4H D3 + D4H D3 + D4H F) 515cjb111 CJB110 2603 h36b DK21 85 51 80 55 80 63 80 59 — — — — (FusionD3 + D4H D3 + D4H D3 + D4H D3 + D4H G) 2603 515 h36b cjb111 86 59 80 6380 51 80 55 — — — — (Fusion D3 + D4H D3 + D4H D3 + D4H D3 + D4H H)cjb111 h36b 2603 515 87 57 80 53 80 61 80 — — — — — (Fusion D2 + D3 +D4H D2 + D3 + D4H D2 + D3 + D4H I) 515 2603 cjb111 *Number indicates SEQID NO:

Thus examples of hybrids of the invention include polypeptidescomprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 83; SEQ: ID NO: 84; SEQ ID NO: 85; SEQ: ID NO: 86; or SEQ IDNO: 87.

The invention provides a polypeptide comprising an amino acid sequencehaving at least i % sequence identity to any one of SEQ ID NOs: 83, 84,85, 86, or 87. The value of i may be selected from 50, 60, 70, 80, 85,90, 95, 96, 97, 98, 99 or more.

In some embodiments, the polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 83; SEQ: ID NO: 84;SEQ: ID NO: 85; SEQ ID NO: 86; or SEQ ID NO: 87, or having at least i %sequence identity to any one of SEQ ID NO: 83; SEQ: ID NO: 84; SEQ: IDNO: 85; SEQ: ID NO: 86; or SEQ ID NO: 87 may comprise a singleN-terminal Methionine residue (i.e. A=Met).

Polypeptides

Polypeptides used with the invention can be prepared in many ways e.g.by chemical synthesis (in whole or in part), by digesting longerpolypeptides using proteases, by translation from RNA, by purificationfrom cell culture (e.g. from recombinant expression), from the organismitself (e.g. after bacterial culture, or direct from patients), etc. Apreferred method for production of peptides <40 amino acids longinvolves in vitro chemical synthesis [5,6]. Solid-phase peptidesynthesis is particularly preferred, such as methods based on tBoc orFmoc [7] chemistry. Enzymatic synthesis [8] may also be used in part orin full. As an alternative to chemical synthesis, biological synthesismay be used e.g. the polypeptides may be produced by translation. Thismay be carried out in vitro or in vivo. Biological methods are ingeneral restricted to the production of polypeptides based on L-aminoacids, but manipulation of translation machinery (e.g. of aminoacyl tRNAmolecules) can be used to allow the introduction of D-amino acids (or ofother non natural amino acids, such as iodotyrosine ormethylphenylalanine, azidohomoalanine, etc.) [9]. Where D-amino acidsare included, however, it is preferred to use chemical synthesis.Polypeptides may have covalent modifications at the C-terminus and/orN-terminus.

Polypeptides can take various forms (e.g. native, fusions, glycosylated,non-glycosylated, lipidated, non-lipidated, phosphorylated,non-phosphorylated, myristoylated, non-myristoylated, monomeric,multimeric, particulate, denatured, etc.).

Polypeptides are preferably provided in purified or substantiallypurified form i.e. substantially free from other polypeptides (e.g. freefrom naturally-occurring polypeptides), particularly from otherpneumococcal or host cell polypeptides, and are generally at least about50% pure (by weight), and usually at least about 90% pure i.e. less thanabout 50%, and more preferably less than about 10% (e.g. 5% or less) ofa composition is made up of other expressed polypeptides.

Polypeptides may be attached to a solid support. Polypeptides maycomprise a detectable label (e.g. a radioactive or fluorescent label, ora biotin label).

The term “polypeptide” refers to amino acid polymers of any length. Thepolymer may be linear or branched, it may comprise modified amino acids,and it may be interrupted by non-amino acids. The terms also encompassan amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.Polypeptides can occur as single chains or associated chains.Polypeptides can be naturally or non-naturally glycosylated (i.e. thepolypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringpolypeptide).

The invention provides a process for producing polypeptides of theinvention, comprising culturing a host cell of to the invention underconditions which induce polypeptide expression. Although expression ofthe polypeptide may take place in a Streptococcus, the invention willusually use a heterologous host for expression. The heterologous hostmay be prokaryotic (e.g. a bacterium) or eukaryotic. It will usually beE. coli, but other suitable hosts include Bacillus subtilis, Vibriocholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica,Neisseria cinerea, Mycobacteria (e.g. M. tuberculosis), yeasts, etc.

The invention also provides a process for producing a polypeptide of theinvention, wherein the polypeptide is synthesised in part or in wholeusing chemical means.

The invention also provides a composition comprising two or morepolypeptides of the invention.

Nucleic Acids

The invention also provides a nucleic acid comprising a nucleotidesequence encoding a hybrid polypeptide of the invention. For example,the invention provides a nucleic acid comprising a nucleotide sequenceencoding a hybrid polypeptide comprising an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 83; SEQ: ID NO: 84; SEQ ID NO:85; SEQ: ID NO: 86; or SEQ ID NO: 87. The invention thus provides anucleic acid comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO:88 (encoding the polypeptide of SEQ ID NO:83),SEQ ID NO: 89 (encoding the polypeptide of SEQ ID NO:84); SEQ ID NO: 90(encoding the polypeptide of SEQ ID NO:85); SEQ ID NO: 91 (encoding thepolypeptide of SEQ ID NO:86); or SEQ ID NO: 92 (encoding the polypeptideof SEQ ID NO:87).

The invention also provides nucleic acid comprising nucleotide sequenceshaving sequence identity to such nucleotide sequences. Such nucleicacids include those using alternative codons to encode the same aminoacid. In particular, nucleic acids may contain alternative codonsoptimised for expression in specific microorganisms. The invention thusprovides a nucleic acid sequence comprising a nucleotide sequenceencoding a hybrid polypeptide comprising an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 83; SEQ: ID NO: 84; SEQ: ID NO:85; SEQ: ID NO: 86; or SEQ ID NO: 87 that has been optimised forexpression in E. coli. The invention thus provides a nucleic acidcomprising a nucleotide sequence selected from the group consisting of:SEQ ID NO:93 (E. coli optimised sequence encoding the polypeptide of SEQID NO:83), SEQ ID NO: 94 (E. coli optimised sequence encoding thepolypeptide of SEQ ID NO:84); SEQ ID NO: 95 (E. coli optimised sequenceencoding the polypeptide of SEQ ID NO:85); SEQ ID NO: 96 (E. colioptimised sequence encoding the polypeptide of SEQ ID NO:86); or SEQ IDNO: 97 (E. coli optimised sequence encoding the polypeptide of SEQ IDNO:87).

The invention also provides nucleic acid which can hybridize to thesenucleic acids. Hybridization reactions can be performed under conditionsof different “stringency”.

Conditions that increase stringency of a hybridization reaction ofwidely known and published in the art. Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C., 55° C. and 68° C.; buffer concentrations of10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mMcitrate buffer) and their equivalents using other buffer systems;formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from5 minutes to 24 hours; 1, 2, or more washing steps; wash incubationtimes of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC,0.1×SSC, or de-ionized water. Hybridization techniques and theiroptimization are well known in the art [e.g. see refs 10 & 223, etc.].

The invention includes nucleic acid comprising sequences complementaryto these sequences (e.g. for antisense or probing, or for use asprimers).

Nucleic acid according to the invention can take various forms (e.g.single-stranded, double-stranded, vectors, primers, probes, labelledetc.). Nucleic acids of the invention may be circular or branched, butwill generally be linear. Unless otherwise specified or required, anyembodiment of the invention that utilizes a nucleic acid may utilizeboth the double-stranded form and each of two complementarysingle-stranded forms which make up the double-stranded form. Primersand probes are generally single-stranded, as are antisense nucleicacids.

Nucleic acids of the invention are preferably provided in purified orsubstantially purified form i.e. substantially free from other nucleicacids (e.g. free from naturally-occurring nucleic acids), particularlyfrom other GBS or host cell nucleic acids, generally being at leastabout 50% pure (by weight), and usually at least about 90% pure. Nucleicacids of the invention are preferably GBS nucleic acids.

Nucleic acids of the invention may be prepared in many ways e.g. bychemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole orin part, by digesting longer nucleic acids using nucleases (e.g.restriction enzymes), by joining shorter nucleic acids or nucleotides(e.g. using ligases or polymerases), from genomic or cDNA libraries,etc.

Nucleic acid of the invention may be attached to a solid support (e.g. abead, plate, filter, film, slide, microarray support, resin, etc.).Nucleic acid of the invention may be labelled e.g. with a radioactive orfluorescent label, or a biotin label. This is particularly useful wherethe nucleic acid is to be used in detection techniques e.g. where thenucleic acid is a primer or as a probe.

The term “nucleic acid” includes in general means a polymeric form ofnucleotides of any length, which contain deoxyribonucleotides,ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNAhybrids. It also includes DNA or RNA analogs, such as those containingmodified backbones (e.g. peptide nucleic acids (PNAs) orphosphorothioates) or modified bases. Thus the invention includes mRNA,tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branchednucleic acids, plasmids, vectors, probes, primers, etc. Where nucleicacid of the invention takes the form of RNA, it may or may not have a 5′cap.

Nucleic acids of the invention may be part of a vector i.e. part of anucleic acid construct designed for transduction/transfection of one ormore cell types. Vectors may be, for example, “cloning vectors” whichare designed for isolation, propagation and replication of insertednucleotides, “expression vectors” which are designed for expression of anucleotide sequence in a host cell, “viral vectors” which is designed toresult in the production of a recombinant virus or virus-like particle,or “shuttle vectors”, which comprise the attributes of more than onetype of vector. Preferred vectors are plasmids. A “host cell” includesan individual cell or cell culture which can be or has been a recipientof exogenous nucleic acid. Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in total DNA complement) to the original parent cell dueto natural, accidental, or deliberate mutation and/or change. Host cellsinclude cells transfected or infected in vivo or in vitro with nucleicacid of the invention.

Where a nucleic acid is DNA, it will be appreciated that “U” in a RNAsequence will be replaced by “T” in the DNA. Similarly, where a nucleicacid is RNA, it will be appreciated that “T” in a DNA sequence will bereplaced by “U” in the RNA.

The term “complement” or “complementary” when used in relation tonucleic acids refers to Watson-Crick base pairing. Thus the complementof C is G, the complement of G is C, the complement of A is T (or U),and the complement of T (or U) is A. It is also possible to use basessuch as I (the purine inosine) e.g. to complement pyrimidines (C or T).

Nucleic acids of the invention can be used, for example: to producepolypeptides in vitro or in vivo; as hybridization probes for thedetection of nucleic acid in biological samples; to generate additionalcopies of the nucleic acids; to generate ribozymes or antisenseoligonucleotides; as single-stranded DNA primers or probes; or astriple-strand forming oligonucleotides.

The invention provides a process for producing nucleic acid of theinvention, wherein the nucleic acid is synthesised in part or in wholeusing chemical means.

The invention provides vectors comprising nucleotide sequences of theinvention (e.g. cloning or expression vectors) and host cellstransformed with such vectors.

Immunogenic Compositions

Mixtures and hybrid polypeptides of the invention are useful as activeingredients in immunogenic compositions. Such immunogenic compositionsmay be useful as vaccines. These vaccines may either be prophylactic(i.e. to prevent infection) or therapeutic (i.e. to treat infection),but will typically be prophylactic.

Compositions may thus be pharmaceutically acceptable. They will usuallyinclude components in addition to the antigens e.g. they typicallyinclude one or more pharmaceutical carrier(s) and/or excipient(s). Athorough discussion of such components is available in reference [218].

Compositions will generally be administered to a mammal in aqueous form.Prior to administration, however, the composition may have been in anon-aqueous form. For instance, although some vaccines are manufacturedin aqueous form, then filled and distributed and administered also inaqueous form, other vaccines are lyophilised during manufacture and arereconstituted into an aqueous form at the time of use. Thus acomposition of the invention may be dried, such as a lyophilisedformulation.

The composition may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free. Vaccines containing no mercury are more preferred.Preservative-free vaccines are particularly preferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml e.g. about 10±2 mg/ml NaCl. Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride,calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.

The composition is preferably sterile. The composition is preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The composition is preferablygluten free.

The composition may include material for a single immunisation, or mayinclude material for multiple immunisations (i.e. a ‘multidose’ kit).The inclusion of a preservative is preferred in multidose arrangements.As an alternative (or in addition) to including a preservative inmultidose compositions, the compositions may be contained in a containerhaving an aseptic adaptor for removal of material.

Human vaccines are typically administered in a dosage volume of about0.5 ml, although a half dose (i.e. about 0.25 ml) may be administered tochildren.

Immunogenic compositions of the invention may also comprise one or moreimmunoregulatory agents. Preferably, one or more of the immunoregulatoryagents include one or more adjuvants. The adjuvants may include a TH1adjuvant and/or a TH2 adjuvant, further discussed below.

Adjuvants which may be used in compositions of the invention include,but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 11], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ [chapter 9 of ref 11]. The degree of crystallinity of analuminium hydroxide adjuvant is reflected by the width of thediffraction band at half height (WHH), with poorly-crystalline particlesshowing greater line broadening due to smaller crystallite sizes. Thesurface area increases as WHH increases, and adjuvants with higher WHHvalues have been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g. as seen in transmission electron micrographs)is typical for aluminium hydroxide adjuvants. The pI of aluminiumhydroxide adjuvants is typically about 11 i.e. the adjuvant itself has apositive surface charge at physiological pH. Adsorptive capacities ofbetween 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate (i.e.aluminium hydroxyphosphate sulfate). They may be obtained byprecipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.) indicates thepresence of structural hydroxyls [ch. 9 of ref. 11].

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate-like morphology as seen intransmission electron micrographs). Typical diameters of the particlesare in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigenadsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mgAl⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of theinvention may contain a buffer (e.g. a phosphate or a histidine or aTris buffer), but this is not always necessary. The suspensions arepreferably sterile and pyrogen-free. A suspension may include freeaqueous phosphate ions e.g. present at a concentration between 1.0 and20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.The suspensions may also comprise sodium chloride.

In one embodiment, an adjuvant component includes a mixture of both analuminium hydroxide and an aluminium phosphate. In this case there maybe more aluminium phosphate than hydroxide e.g. a weight ratio of atleast 2:1 e.g. ≥5:1, ≥6:1, ≥7:1, ≥8:1, ≥9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g. ≤5 mg/ml, ≤4 mg/ml, ≤3mg/ml, ≤2 mg/ml, ≤1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of <0.85 mg/dose is preferred.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59® [Chapter 10 of ref. 11;see also ref 12] (5% squalene, 0.5% TWEEN® 80, and 0.5% SPAN® 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

Various suitable oil-in-water emulsions are known, and they typicallyinclude at least one oil and at least one surfactant, with the oil(s)and surfactant(s) being biodegradable (metabolisable) and biocompatible.The oil droplets in the emulsion are generally less than 5 μm indiameter, and advantageously the emulsion comprises oil droplets with asub-micron diameter, with these small sizes being achieved with amicrofluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm are preferred as they can be subjected to filtersterilization.

The invention can be used with oils such as those from an animal (suchas fish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Otherpreferred oils are the tocopherols (see below). Oil in water emulsionscomprising squalene are particularly preferred. Mixtures of oils can beused.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (TRITON® X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as BRIJ®surfactants), such as triethyleneglycol monolauryl ether (BRIJ® 30); andsorbitan esters (commonly known as the SPANs), such as sorbitantrioleate (SPAN® 85) and sorbitan monolaurate. Preferred surfactants forincluding in the emulsion are TWEEN® 80 (polyoxyethylene sorbitanmonooleate), SPAN® 85 (sorbitan trioleate), lecithin and TRITON® X-100.As mentioned above, detergents such as Tween 80 may contribute to thethermal stability seen in the examples below.

Mixtures of surfactants can be used e.g. TWEEN® 80/SPAN® 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (TWEEN® 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (TRITON® X-100) is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as TWEEN® 80) 0.01 to 1%, in particular about0.1%; octyl- or nonylphenoxy polyoxyethanols (such as TRITON® X-100, orother detergents in the TRITON® series) 0.001 to 0.1%, in particular0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%,preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% SPAN® 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% SPAN® 85. This adjuvant is known as ‘MF59®’ [13-15],        as described in more detail in Chapter 10 of ref 16 and chapter        12 of ref. 17. The MF59® emulsion advantageously includes        citrate ions e.g. 10 mM sodium citrate buffer.    -   An emulsion comprising squalene, an α-tocopherol, and        polysorbate 80. These emulsions may have from 2 to 10% squalene,        from 2 to 10% tocopherol and from 0.3 to 3% TWEEN® 80, and the        weight ratio of squalene:tocopherol is preferably ≤1 (e.g. 0.90)        as this provides a more stable emulsion. Squalene and TWEEN® 80        may be present volume ratio of about 5:2, or at a weight ratio        of about 11:5. One such emulsion can be made by dissolving        TWEEN® 80 in PBS to give a 2% solution, then mixing 90 ml of        this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml        squalene), then microfluidising the mixture. The resulting        emulsion may have submicron oil droplets e.g. with an average        diameter of between 100 and 250 nm, preferably about 180 nm.    -   An emulsion of squalene, a tocopherol, and a TRITON® detergent        (e.g. TRITON® X-100). The emulsion may also include a 3d-MPL®        (see below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        TRITON® detergent (e.g. TRITON® X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml TRITON® X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“PLURONIC® L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [18] (0.05-1% Thr-MDP, 5%        squalane, 2.5% PLURONIC® L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [19]        (5% squalene, 1.25% PLURONIC® L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘SPAN® 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [20]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. Such emulsions        may be lyophilized.    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 21, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, TWEEN® 80 or SPAN® 80). Additives may be included,        such as QuilA saponin, cholesterol, a saponin-lipophile        conjugate (such as GPI-0100, described in reference 22, produced        by addition of aliphatic amine to desacylsaponin via the        carboxyl group of glucuronic acid), dimethyidioctadecylammonium        bromide and/or N,N-dioctadecyl-N,N-bis        (2-hydroxyethyl)propanediamine.    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [23].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [23].    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [24].

Antigens and adjuvants in a composition will typically be in admixtureat the time of delivery to a patient. The emulsions may be mixed withantigen during manufacture, or extemporaneously, at the time ofdelivery. Thus the adjuvant and antigen may be kept separately in apackaged or distributed vaccine, ready for final formulation at the timeof use. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g. between 5:1 and 1:5) but isgenerally about 1:1.

C. Saponin Formulations [Chapter 22 of Ref 11]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterogeneous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref 25.Saponin formulations may also comprise a sterol, such as cholesterol[26].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs) [chapter 23 of ref11]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA & QHC. ISCOMs are further described in refs. 26-28. Optionally, theISCOMS may be devoid of additional detergent [29].

A review of the development of saponin based adjuvants can be found inrefs. 30 & 31.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 32-37. Virosomes are discussed further in, for example, ref 38

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL®) and3-O-deacylated MPL® (3dMPL®). 3dMPL® is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref 39. Such “small particles” of 3dMPL® are small enoughto be sterile filtered through a 0.22 μm membrane [39]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [40,41].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 42 & 43.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 44, 45 and 46 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 47-52.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [53]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 54-56. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 53 & 57-59.

A particularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC-31™ [60]. Thus an adjuvant used with theinvention may comprise a mixture of (i) an oligonucleotide (e.g. between15-40 nucleotides) including at least one (and preferably multiple) CpImotifs (i.e. a cytosine linked to an inosine to form a dinucleotide),and (ii) a polycationic polymer, such as an oligopeptide (e.g. between5-20 amino acids) including at least one (and preferably multiple)Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be adeoxynucleotide comprising 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID NO: 98).The polycationic polymer may be a peptide comprising 11-mer amino acidsequence KLKLLLLLKLK (SEQ ID NO:99).

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 61 and as parenteraladjuvants in ref 62. The toxin or toxoid is preferably in the form of aholotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 63-70. A useful CT mutant is or CT-E29H[71]. Numerical reference for amino acid substitutions is preferablybased on the alignments of the A and B subunits of ADP-ribosylatingtoxins set forth in ref 72, specifically incorporated herein byreference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [73], etc.) [74], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor. Apreferred immunomodulator is IL-12.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [75] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [76].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref 11)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 77-79.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [80]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [81] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [82]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 83 and 84.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 85 and 86.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [87]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL®) [88]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL®)+a cholesterol; (4) a saponin (e.g. QS21)+3dMPL®+IL-12 (optionally+a sterol) [89]; (5) combinations of 3dMPL® with, forexample, QS21 and/or oil-in-water emulsions [90]; (6) SAF, containing10% squalene, 0.4% TWEEN® 80, 5% PLURONIC® block polymer L121, andthr-MDP, either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion. (7) Ribi adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% TWEEN® 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL®), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL® +CWS (DETOX™); and (8) one or moremineral salts (such as an aluminum salt)+a non-toxic derivative of LPS(such as 3 dMPL®).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 11.

The use of an aluminium hydroxide and/or aluminium phosphate adjuvant isuseful, particularly in children, and antigens are generally adsorbed tothese salts. Squalene-in-water emulsions are also preferred,particularly in the elderly. Useful adjuvant combinations includecombinations of Th1 and Th2 adjuvants such as CpG & alum or resiquimod &alum. A combination of aluminium phosphate and 3dMPL may be used.

The compositions of the invention may elicit both a cell mediated immuneresponse as well as a humoral immune response.

Two types of T cells, CD4 and CD8 cells, are generally thought necessaryto initiate and/or enhance cell mediated immunity and humoral immunity.CD8 T cells can express a CD8 co-receptor and are commonly referred toas Cytotoxic T lymphocytes (CTLs). CD8 T cells are able to recognized orinteract with antigens displayed on MHC Class I molecules.

CD4 T cells can express a CD4 co-receptor and are commonly referred toas T helper cells. CD4 T cells are able to recognize antigenic peptidesbound to MHC class II molecules. Upon interaction with a MHC class IImolecule, the CD4 cells can secrete factors such as cytokines. Thesesecreted cytokines can activate B cells, cytotoxic T cells, macrophages,and other cells that participate in an immune response. Helper T cellsor CD4+cells can be further divided into two functionally distinctsubsets: TH1 phenotype and TH2 phenotypes which differ in their cytokineand effector function.

Activated TH1 cells enhance cellular immunity (including an increase inantigen-specific CTL production) and are therefore of particular valuein responding to intracellular infections. Activated TH1 cells maysecrete one or more of IL-2, IFN-γ, and TNF-β. A TH1 immune response mayresult in local inflammatory reactions by activating macrophages, NK(natural killer) cells, and CD8 cytotoxic T cells (CTLs). A TH1 immuneresponse may also act to expand the immune response by stimulatinggrowth of B and T cells with IL-12. TH1 stimulated B cells may secreteIgG2a.

Activated TH2 cells enhance antibody production and are therefore ofvalue in responding to extracellular infections. Activated TH2 cells maysecrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immuneresponse may result in the production of IgG1, IgE, IgA and memory Bcells for future protection.

An enhanced immune response may include one or more of an enhanced TH1immune response and a TH2 immune response.

A TH1 immune response may include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a TH1 immuneresponse (such as IL-2, IFN-γ, and TNF-β), an increase in activatedmacrophages, an increase in NK activity, or an increase in theproduction of IgG2a. Preferably, the enhanced TH1 immune response willinclude an increase in IgG2a production.

A TH1 immune response may be elicited using a TH1 adjuvant. A TH1adjuvant will generally elicit increased levels of IgG2a productionrelative to immunization of the antigen without adjuvant. TH1 adjuvantssuitable for use in the invention may include for example saponinformulations, virosomes and virus like particles, non-toxic derivativesof enterobacterial lipopolysaccharide (LPS), immunostimulatoryoligonucleotides. Immunostimulatory oligonucleotides, such asoligonucleotides containing a CpG motif, are preferred TH1 adjuvants foruse in the invention.

A TH2 immune response may include one or more of an increase in one ormore of the cytokines associated with a TH2 immune response (such asIL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1,IgE, IgA and memory B cells. Preferably, the enhanced TH2 immuneresponse will include an increase in IgG1 production.

A TH2 immune response may be elicited using a TH2 adjuvant. A TH2adjuvant will generally elicit increased levels of IgG1 productionrelative to immunization of the antigen without adjuvant. TH2 adjuvantssuitable for use in the invention include, for example, mineralcontaining compositions, oil-emulsions, and ADP-ribosylating toxins anddetoxified derivatives thereof. Mineral containing compositions, such asaluminium salts are preferred TH2 adjuvants for use in the invention.

A composition may include a combination of a TH1 adjuvant and a TH2adjuvant. Preferably, such a composition elicits an enhanced TH1 and anenhanced TH2 response, i.e., an increase in the production of both IgG1and IgG2a production relative to immunization without an adjuvant. Stillmore preferably, the composition comprising a combination of a TH1 and aTH2 adjuvant elicits an increased TH1 and/or an increased TH2 immuneresponse relative to immunization with a single adjuvant (i.e., relativeto immunization with a TH1 adjuvant alone or immunization with a TH2adjuvant alone).

The immune response may be one or both of a TH1 immune response and aTH2 response. Preferably, immune response provides for one or both of anenhanced TH1 response and an enhanced TH2 response.

The enhanced immune response may be one or both of a systemic and amucosal immune response. Preferably, the immune response provides forone or both of an enhanced systemic and an enhanced mucosal immuneresponse. Preferably the mucosal immune response is a TH2 immuneresponse. Preferably, the mucosal immune response includes an increasein the production of IgA.

Streptococcal infections can affect various areas of the body and so thecompositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g.

a lyophilised composition or a spray-freeze dried composition). Thecomposition may be prepared for topical administration e.g. as anointment, cream or powder. The composition may be prepared for oraladministration e.g. as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops.The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a patient.Such kits may comprise one or more antigens in liquid form and one ormore lyophilised antigens.

Where a composition is to be prepared extemporaneously prior to use(e.g. where a component is presented in lyophilised form) and ispresented as a kit, the kit may comprise two vials, or it may compriseone ready-filled syringe and one vial, with the contents of the syringebeing used to reactivate the contents of the vial prior to injection.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s), as well as any other components, asneeded. By ‘immunologically effective amount’, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

Nucleic Acid Immunisation

The immunogenic compositions described above include polypeptideantigens from GBS. In all cases, however, the polypeptide antigens canbe replaced by nucleic acids (typically DNA) encoding thosepolypeptides, to give compositions, methods and uses based on nucleicacid immunisation [91 to 98].

The nucleic acid encoding the immunogen is expressed in vivo afterdelivery to a patient and the expressed immunogen then stimulates theimmune system. The active ingredient will typically take the form of anucleic acid vector comprising: (i) a promoter; (ii) a sequence encodingthe immunogen, operably linked to the promoter; and optionally (iii) aselectable marker. Preferred vectors may further comprise (iv) an originof replication; and (v) a transcription terminator downstream of andoperably linked to (ii). In general, (i) & (v) will be eukaryotic and(iii) & (iv) will be prokaryotic.

Preferred promoters are viral promoters e.g. from cytomegalovirus (CMV).The vector may also include transcriptional regulatory sequences (e.g.enhancers) in addition to the promoter and which interact functionallywith the promoter. Preferred vectors include the immediate-early CMVenhancer/promoter, and more preferred vectors also include CMV intron A.The promoter is operably linked to a downstream sequence encoding animmunogen, such that expression of the immunogen-encoding sequence isunder the promoter's control.

Where a marker is used, it preferably functions in a microbial host(e.g. in a prokaryote, in a bacteria, in a yeast). The marker ispreferably a prokaryotic selectable marker (e.g. transcribed under thecontrol of a prokaryotic promoter). For convenience, typical markers areantibiotic resistance genes.

The vector is preferably an autonomously replicating episomal orextrachromosomal vector, such as a plasmid.

The vector preferably comprises an origin of replication. It ispreferred that the origin of replication is active in prokaryotes butnot in eukaryotes.

Preferred vectors thus include a prokaryotic marker for selection of thevector, a prokaryotic origin of replication, but a eukaryotic promoterfor driving transcription of the immunogen-encoding sequence. Thevectors will therefore (a) be amplified and selected in prokaryotichosts without polypeptide expression, but (b) be expressed in eukaryotichosts without being amplified. This arrangement is ideal for nucleicacid immunization vectors.

The vector may comprise a eukaryotic transcriptional terminator sequencedownstream of the coding sequence. This can enhance transcriptionlevels. Where the coding sequence does not have its own, the vectorpreferably comprises a polyadenylation sequence. A preferredpolyadenylation sequence is from bovine growth hormone.

The vector may comprise a multiple cloning site

In addition to sequences encoding the immunogen and a marker, the vectormay comprise a second eukaryotic coding sequence. The vector may alsocomprise an IRES upstream of said second sequence in order to permittranslation of a second eukaryotic polypeptide from the same transcriptas the immunogen. Alternatively, the immunogen-coding sequence may bedownstream of an IRES.

The vector may comprise unmethylated CpG motifs e.g. unmethylated DNAsequences which have in common a cytosine preceding a guanosine, flankedby two 5′ purines and two 3′ pyrimidines. In their unmethylated formthese DNA motifs have been demonstrated to be potent stimulators ofseveral types of immune cell.

Vectors may be delivered in a targeted way. Receptor-mediated DNAdelivery techniques are described in, for example, references 99 to 104.Therapeutic compositions containing a nucleic acid are administered in arange of about 100 ng to about 200 mg of DNA for local administration ina gene therapy protocol. Concentration ranges of about 500 ng to about50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about20 μg to about 100 μg of DNA can also be used during a gene therapyprotocol. Factors such as method of action (e.g. for enhancing orinhibiting levels of the encoded gene product) and efficacy oftransformation and expression are considerations which will affect thedosage required for ultimate efficacy. Where greater expression isdesired over a larger area of tissue, larger amounts of vector or thesame amounts re-administered in a successive protocol ofadministrations, or several administrations to different adjacent orclose tissue portions may be required to effect a positive therapeuticoutcome. In all cases, routine experimentation in clinical trials willdetermine specific ranges for optimal therapeutic effect.

Vectors can be delivered using gene delivery vehicles. The gene deliveryvehicle can be of viral or non-viral origin (see generally references105 to 108).

Viral-based vectors for delivery of a desired nucleic acid andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (e.g. references 109 to 119), alphavirus-based vectors(e.g. Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCCVR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelanequine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532); hybrids or chimeras of these viruses may also be used),poxvirus vectors (e.g. vaccinia, fowlpox, canarypox, modified vacciniaAnkara, etc.), adenovirus vectors, and adeno-associated virus (AAV)vectors (e.g. see refs. 120 to 125). Administration of DNA linked tokilled adenovirus [126] can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone [e.g. 126], ligand-linked DNA [127], eukaryoticcell delivery vehicles cells [e.g. refs. 128 to 132] and nucleic chargeneutralization or fusion with cell membranes. Naked DNA can also beemployed. Exemplary naked DNA introduction methods are described inrefs. 133 and 134. Liposomes (e.g. immunoliposomes) that can act as genedelivery vehicles are described in refs. 135 to 139. Additionalapproaches are described in references 140 & 141.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in ref 141. Moreover, the codingsequence and the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials or use of ionizingradiation [e.g. refs. 142 & 143]. Other conventional methods for genedelivery that can be used for delivery of the coding sequence include,for example, use of hand-held gene transfer particle gun [144] or use ofionizing radiation for activating transferred genes [142 & 143].

Delivery of DNA using PLG {poly(lactide-co-glycolide)} microparticles isa particularly preferred method e.g. by adsorption to themicroparticles, which are optionally treated to have anegatively-charged surface (e.g. treated with SDS) or apositively-charged surface (e.g. treated with a cationic detergent, suchas CTAB).

Methods of Treatment, and Administration of the Vaccine

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of animmunogenic composition of the invention. The immune response ispreferably protective and preferably involves antibodies and/orcell-mediated immunity. The method may raise a booster response.

The invention also provides at least two different GBS59 clades forcombined use as a medicament e.g. for use in raising an immune responsein a mammal.

The invention also provides the use of at least two different GBS59clades in the manufacture of a medicament for raising an immune responsein a mammal.

By raising an immune response in the mammal by these uses and methods,the mammal can be protected against disease and/or infection caused byGBS e.g. against meningitis.

The invention also provides a delivery device pre-filled with animmunogenic composition of the invention.

The mammal is preferably a human. The human may be a child (e.g. atoddler or infant), a teenager or an adult. A vaccine intended forchildren may also be administered to adults e.g. to assess safety,dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pneumococcal infection after administration of thecompositions of the invention. One way of checking efficacy ofprophylactic treatment involves testing post-immunisation sera instandard tests; for example, sera can be tested in an opsonophagocytickilling assay (OPKA), with the ability to opsonise bacteria indicatingprotective efficacy. Another way of checking efficacy of prophylactictreatment involves post-immunisation challenge in an animal model of GBSinfection, e.g., guinea pigs or mice. One such model is described inreference 145. Another way of assessing the immunogenicity of thecompositions of the present invention is to express the polypeptidesrecombinantly for screening patient sera or mucosal secretions byimmunoblot and/or microarrays. A positive reaction between thepolypeptide and the patient sample indicates that the patient hasmounted an immune response to the polypeptide in question. This methodmay also be used to identify immunodominant antigens and/or epitopeswithin antigens.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or mucosally, such as byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal ortranscutaneous, intranasal, ocular, aural, pulmonary or other mucosaladministration.

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Preferably the enhanced systemic and/or mucosal immunity is reflected inan enhanced TH1 and/or TH2 immune response. Preferably, the enhancedimmune response includes an increase in the production of IgG1 and/orIgG2a and/or IgA.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. In general, the vaccinesmay be used to treat pregnant women and adolescents. The vaccines arenot suitable solely for these groups, however, and may be used moregenerally in a population.

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially at the same time as arubella vaccine, a varicella vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, an inactivated poliovirusvaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine(such as a tetravalent A-C-W135-Y vaccine), a respiratory syncytialvirus vaccine, an human papillomavirus vaccine, an influenza virusvaccines (including a pandemic influenza virus vaccine) etc.

Vaccines of the invention may also be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a (3R,4R,5S)-4-acetylamino-5-amino-3 (1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-l-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

Combinations

A composition useful for immunisation comprises at least two GBS59clades, either as a hybrid polypeptide or as separate polypeptides. Inaddition, a composition may include:

(i) one or more further polypeptides that elicit antibody responsesagainst GBS proteins, particularly against GBS proteins other thanGBS59; (ii) a capsular saccharide from GBS; and/or (iii) one or morefurther immunogens that elicit antibody responses that recogniseepitopes on non-GBS organisms.

Combinations with Further Polypeptide Antigens [146]

GBS polypeptides from two or more clades may be combined with one ormore (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10) polypeptide antigensselected from the group consisting of: (1) a (GBS80) antigen; (2) aGBS67 antigen; (3) a GBS1523 antigen; (4) a GBS 104 antigen; (5) aGBS1524 antigen; (6) a GBS3 antigen; (7) a SAN1483 antigen; (8) a GBS147antigen; (9) a GBS328 antigen; and/or (10) a GBS84 antigen.

These further antigens may be added as separate polypeptides. As analternative, they may be added as hybrids e.g. a GBS80-GBS1523 hybrid.As a further alternative, they may be fused to a GBS59 polypeptidesequence to provide a hybrid polypeptide e.g. a GBS59-GBS80 hybrid.

Any of these combinations may also include one or more GBS capsularsaccharide(s), which will typically be conjugated to carrier protein(s).Further information about such saccharides and conjugation is providedbelow.

GBS80

The original ‘GBS80’ (SAG0645) sequence was annotated in reference 147as a cell wall surface anchor family protein (see GI: 22533660). Forreference purposes, the amino acid sequence of full length GBS80 asfound in the 2603 strain is given as SEQ ID NO: 177 herein. PreferredGBS80 polypeptides for use with the invention comprise an amino acidsequence: (a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO:177; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 177, wherein ‘n’ is 7 or more(e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200, 250 or more). These GBS80 proteins include variants ofSEQ ID NO: 177.

Preferred fragments of (b) comprise an epitope from SEQ ID NO: 177.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 177 while retaining at least oneepitope of SEQ ID NO: 177. Other fragments omit one or more proteindomains.

Wild-type GBS80 contains a N-terminal leader or signal sequence regionat amino acids 1-37 of SEQ ID NO:177. One or more amino acids from theleader or signal sequence region of GBS80 can be removed, e.g. SEQ IDNO:178. The wild-type sequence also contains a C-terminal transmembraneregion at amino acids 526-543 of SEQ ID NO: 177. One or more amino acidsfrom the transmembrane region and/or a cytoplasmic region may beremoved, e.g. SEQ ID NO:179. Wild-type GBS80 contains an amino acidmotif indicative of a cell wall anchor at amino acids 521-525 of SEQ IDNO:177. In some recombinant host cell systems it may be useful to removethis motif to facilitate secretion of a recombinant GBS80 polypeptidefrom the host cell. Thus the transmembrane and/or cytoplasmic regionsand the cell wall anchor motif may be removed from GBS80, e.g. SEQ IDNO:180. Alternatively, in some recombinant host cell systems it may beuseful to use the cell wall anchor motif to anchor the recombinantlyexpressed polypeptide to the cell wall. The extracellular domain of theexpressed polypeptide may be cleaved during purification or therecombinant polypeptide may be left attached to either inactivated hostcells or cell membranes in the final composition, e.g. SEQ ID NO:181. Aparticularly immunogenic fragment of wild-type GBS80 is located towardsthe N-terminus of the polypeptide, and is SEQ ID NO:182.

GBS67

The original ‘GBS67’ (SAG1408) sequence was annotated in reference 147as a cell wall surface anchor family protein (see GI: 22534437). Forreference purposes, the amino acid sequence of full length GBS67 asfound in the 2603 strain is given as SEQ ID NO: 183 herein. PreferredGBS67 polypeptides for use with the invention comprise an amino acidsequence: (a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 183; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 183, wherein ‘n’ is 7 or more(e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200, 250 or more). These GBS67 proteins include variants ofSEQ ID NO: 183. Preferred fragments of (b) comprise an epitope from SEQID NO: 183. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 183 while retaining atleast one epitope of SEQ ID NO: 183. Other fragments omit one or moreprotein domains.

Wild-type GBS67 contains a C-terminus transmembrane region which may beremoved e.g. to give SEQ ID NO: 184. It also contains amino acid motifsindicative of a cell wall anchor (LPXTG, SEQ ID NO:272; and IPMTG, SEQID NO:273). In some recombinant host cell systems, it may be preferableto remove this motif to facilitate secretion from the host cell.Accordingly, in one preferred fragment of GBS67 for use in theinvention, the transmembrane and the cell wall anchor motif are removed(SEQ ID NO:185). Alternatively, in some recombinant host cell systems,it may be preferable to use the cell wall anchor motif to anchor therecombinantly expressed polypeptide to the cell wall. The extracellulardomain of the expressed polypeptide may be cleaved during purificationor the recombinant polypeptide may be left attached to eitherinactivated host cells or cell membranes in the final composition.

Three pilin motifs, containing conserved lysine residues have beenidentified in GBS67. Conserved lysine residues are at amino acidresidues 478 and 488, at amino acid residues 340 and 342, and at aminoacid residues 703 and 717. The pilin sequences, in particular theconserved lysine residues, are thought to be important for the formationof oligomeric, pilus-like structures of GBS67. Preferred fragments ofGBS67 include at least one conserved lysine residue. Two E boxescontaining conserved glutamic residues have also been identified inGBS67. Preferred fragments of GBS 67 include at least one conservedglutamic acid residue. GBS67 contains several regions predicted to formalpha helical structures. Such alpha helical regions are likely to formcoiled-coil structures and may be involved in oligomerization of GBS67.GBS67 also contains a region which is homologous to the Cna_B domain ofthe S. aureus collagen-binding surface protein (pfam05738). This mayform a beta sandwich structure. GBS67 contains a region which ishomologous to a von Willebrand factor (vWF) type A domain which may alsobe retained in the fragments of GBS67.

Particularly immunogenic fragments of wild-type GBS67 from the 2603strain are located towards the N-terminus of the polypeptide, and areSEQ ID NO:186 and SEQ ID NO:187.

A variant of GBS67 (SAI1512) exists in strain H36B. This variant ‘GBS67’(SAG1408) sequence was annotated in reference 148 as a cell wall surfaceanchor family protein (see GI: 77405751). For reference purposes, theamino acid sequence of full length GBS67 as found in the H36B strain isgiven as SEQ ID NO: 188 herein. Preferred GBS67 polypeptides for usewith the invention comprise an amino acid sequence: (a) having 60% ormore identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 188; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 188, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments of (b) comprise an epitope from SEQ ID NO: 188.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 188 while retaining at least oneepitope of SEQ ID NO: 188. Other fragments omit one or more proteindomains.

The invention includes the use of fragments of GBS67 from the H36Bstrain that are analogous to fragments of GBS67 from the 2603 straindiscussed in detail above, e.g. lacking the C-terminus transmembraneregion, lacking the transmembrane region and/or cell wall anchor motif(LPXTG, SEQ ID NO:272; and IPMTG, SEQ ID NO:273), containing conservedpilin motifs or lysine residues within the pilin motifs, containingconserved glutamic acid residues, alpha helical regions, the Cna_Bdomain and/or the (vWF) type A domain. Particularly immunogenicfragments of wild-type GBS67 from the H36B strain are located towardsthe N-terminus of the polypeptide, and are SEQ ID NO:189 and SEQ IDNO:190.

Variants of GBS67 also exist in strains CJB111, 515, NEM316, DK21 andCJB110. For reference purposes, the amino acid sequences of full lengthGBS67 as found in the CJB111, 515, NEM316, DK21 and CJB110 strains aregiven as SEQ ID NOS: 191, 194, 197, 200 and 203 herein. Preferred GBS67polypeptides for use with the invention comprise an amino acid sequence:(a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ IDNOS: 191, 192, 193, 194 or 195; and/or (b) comprising a fragment of atleast ‘n’ consecutive amino acids of SEQ ID NOS: 191, 194, 197, 200 and203, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferredfragments of (b) comprise an epitope from SEQ ID NOS: 191, 194, 197, 200or 203. Other preferred fragments lack one or more amino acids (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NOS: 191, 194, 197, 200 or203 while retaining at least one epitope of SEQ ID NOS: 191, 194, 197,200 or 203. Other fragments omit one or more protein domains.

The invention includes the use of fragments of GBS67 from the CJB111,515, NEM316, DK21 and CJB110 strains that are analogous to fragments ofGBS67 from the 2603 strain discussed in detail above, e.g. lacking theC-terminus transmembrane region, lacking the transmembrane region and/orcell wall anchor motif (LPXTG, SEQ ID NO:272; and IPMTG, SEQ ID NO:273),containing conserved pilin motifs or lysine residues within the pilinmotifs, containing conserved glutamic acid residues, alpha helicalregions, the Cna_B domain and/or the (vWF) type A domain. Particularlyimmunogenic fragments of wild-type GBS67 from the CJB111, 515, NEM316,DK21 and CJB110 are located towards the N-terminus of the polypeptide,and are SEQ ID NO:192 and SEQ ID NO:193 (CJB111), SEQ ID NO:195 and SEQID NO:196 (515), SEQ ID NO:198 and SEQ ID NO:199 (NEM316), SEQ ID NO:201and SEQ ID NO:202 (DK21), and SEQ ID NO:204 and SEQ ID NO:205 (CJB110).

GBS1523

The original ‘GBS1523’ (SAN1518; SpbI) sequence was annotated inreference 148 as a cell wall surface anchor family protein (see GI:77408651). For reference purposes, the amino acid sequence of fulllength GBS1523 as found in the COH1 strain is given as SEQ ID NO: 206herein. Preferred GBS1523 polypeptides for use with the inventioncomprise an amino acid sequence: (a) having 60% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more) to SEQ ID NO: 206; and/or (b) comprising afragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 206,wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These GBS1523proteins include variants of SEQ ID NO: 206. Preferred fragments of (b)comprise an epitope from SEQ ID NO: 206. Other preferred fragments lackone or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQID NO: 206 while retaining at least one epitope of SEQ ID NO: 206. Otherfragments omit one or more protein domains.

Wild-type GBS1523 contains a N-terminal leader or signal sequence regionat amino acids 1 to 29 of SEQ ID NO:206 which may be removed infragments, e.g. SEQ ID NO:207. The wild-type sequence contains an aminoacid motif indicative of a cell wall anchor (LPSTG) at amino acids468-472 of SEQ ID NO:206. In some recombinant host cell systems, it maybe preferable to remove this motif to facilitate secretion of arecombinant polypeptide from the cell. Alternatively, it may bepreferable to use the cell wall anchor motif to anchor the recombinantlyexpressed polypeptide to the cell wall. The extracellular domain of theexpressed polypeptide may be cleaved during purification or therecombinant polypeptide may be left attached to either inactivated hostcells or cell membranes in the final composition. An E box containing aconserved glutamic residue has also been identified at amino acids419-429 of SEQ ID NO:206, with a conserved glutamic acid at residue 423.The E box motif may be important for the formation of oligomericpilus-like structures, and so useful fragments of GBS1523 may includethe conserved glutamic acid residue. A mutant of GBS1523 has beenidentified in which the glutamine (Q) at position 41 of SEQ ID NO:206 issubstituted for a lysine (K), as a result of a mutation of a codon inthe encoding nucleotide sequence from CAA to AAA. This substitution maybe present in the GBS1523 sequences and GBS1523 fragments (e.g. SEQ IDNO:208).

Where the compositions include both GBS80 and GBS1523, a hybridpolypeptide may be used. Examples of GBS80-GBS1523 hybrids are found inreference 149 and include the polypeptides of SEQ ID NOS: 209-212.

GBS104

The original ‘GBS104’ (SAG0649) sequence was annotated in reference 147as ‘a cell wall surface anchor family protein’ (see GI: 22533664). Forreference purposes, the amino acid sequence of full length GBS104 asfound in the 2603 strain is given as SEQ ID NO: 213 herein. PreferredGBS104 polypeptides for use with the invention comprise an amino acidsequence: (a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 213; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 213, wherein ‘n’ is 7 or more(e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200, 250 or more). These GBS104 proteins include variants ofSEQ ID NO: 213. Preferred fragments of (b) comprise an epitope from SEQID NO: 213. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 213 while retaining atleast one epitope of SEQ ID NO: 213. Other fragments omit one or moreprotein domains.

GBS1524

For reference purposes, the amino acid sequence of full length GBS1524is given as SEQ ID NO: 214 herein. Preferred GBS1524 polypeptides foruse with the invention comprise an amino acid sequence: (a) having 60%or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 214; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 214, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). TheseGBS1524 proteins include variants of SEQ ID NO: 214. Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 214. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 214 while retaining at least one epitope of SEQID NO: 214. Other fragments omit one or more protein domains.

GBS3

The original ‘GBS3’ (SAG2603; BibA) sequence was annotated in reference147 as ‘a pathogenicity protein’ (see GI:22535109). For referencepurposes, the amino acid sequence of full length GBS3 as found in the2603 strain is given as SEQ ID NO: 215 herein. Preferred GBS3polypeptides for use with the invention comprise an amino acid sequence:(a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ IDNO: 215; and/or (b) comprising a fragment of at least ‘n’ consecutiveamino acids of SEQ ID NO: 215, wherein ‘n’ is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250or more). These GBS3 proteins include variants of SEQ ID NO: 215.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 215.

Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 215 while retaining at least oneepitope of SEQ ID NO: 215. Other fragments omit one or more proteindomains.

Wild-type GBS3 contains a N-terminal leader or signal sequence region atamino acids 1 to 36 of SEQ ID NO:215 which may be removed in fragments,e.g. SEQ ID NO:216.GBS3 also contains an amino acid motif indicative ofa cell wall anchor (LPXTG, SEQ ID NO:272), a transmembrane region andcytoplasmic domains (see reference 150). The leader or signal sequenceregion, the transmembrane and cytoplasmic domains, and the cell wallanchor motif may all be removed from GBS3 to leave a fragment comprisingthe coiled-coil and proline-rich segments as set forth below (SEQ IDNO:217). Alternative fragments of GBS3 may comprise: the signal sequenceregion and coiled coil segment (SEQ ID NO:218); the coiled coil segment(SEQ ID NO:219); or the signal sequence region, coiled coil segment, andproline-rich segment (SEQ ID NO:220).

Variants of GBS3 exist in the 515 strain (SAL2118), CJB111 strain(SAM1974) and COH1 strain (SAN2207). Reference amino acid sequences forfull-length GBS3 in the 515 strain, the CJB111 strain and the COH1strain are given herein as SEQ ID NO: 221, SEQ ID NO:222 and SEQ IDNO:223 respectively. Thus, GBS3 polypeptides for use with the inventionmay also comprise an amino acid sequence: (a) having 60% or moreidentity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 221 SEQ ID NO:222or SEQ ID NO:223; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 221, SEQ ID NO:222 or SEQ IDNO:223, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These GBS3proteins include variants of SEQ ID NO: 221, SEQ ID NO:222 or SEQ IDNO:223. Preferred fragments of (b) comprise an epitope from SEQ ID NO:221, SEQ ID NO:222 or SEQ IO NO:223. Other preferred fragments lack oneor more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ IDNO: 221, SEQ ID NO:222 or SEQ ID NO:223while retaining at least oneepitope of SEQ ID NO: 221, SEQ ID NO:222 or SEQ ID NO:223. Otherfragments omit one or more protein domains.

The invention includes the use of fragments of GBS3 from the 515, cjb111and cohl strains that are analogous to fragments of GBS3 from the 2603strain discussed in detail above, e.g. lacking the N-terminal leader orsignal sequence region; comprising the coiled-coil and proline-richsegments; commising the signal sequence region and coiled coil segment;comprising the coiled coil segment; or comprising the signal sequenceregion, coiled coil segment, and proline-rich segment.

SAN1485

The original ‘SAN1485’ sequence was annotated in reference 148 as ‘ cellwall surface anchor family protein ’ (see GI: 77408233). For referencepurposes, the amino acid sequence of full length SAN1485 as found in theCOH1 strain is given as SEQ ID NO: 224 herein. Preferred SAN1485polypeptides for use with the invention comprise an amino acid sequence:(a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ IDNO: 224; and/or (b) comprising a fragment of at least ‘n’ consecutiveamino acids of SEQ ID NO: 224, wherein ‘n’ is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250or more). These SAN1485 proteins include variants of SEQ ID NO: 224.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 224.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 224 while retaining at least oneepitope of SEQ ID NO: 224. Other fragments omit one or more proteindomains.

GBS147

The original ‘GBS147’ (SAG0416) sequence was annotated in reference 147as ‘a putative protease’ (see GI:22533435). For reference purposes, theamino acid sequence of full length GBS147 as found in the 2603 strain isgiven as SEQ ID NO: 225 herein. Preferred GBS147 polypeptides for usewith the invention comprise an amino acid sequence: (a) having 60% ormore identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 225 and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 225, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). TheseGBS147 proteins include variants of SEQ ID NO: 225. Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 225. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 225 while retaining at least one epitope of SEQID NO: 225.

GBS328

The original ‘GBS328’ (SAG1333) sequence was annotated in reference 147as ‘ 5’-nucleotidase family protein' (see GI: 22534359). For referencepurposes, the amino acid sequence of full length GBS328 as found in the2603 strain is given as SEQ ID NO:226 herein. Preferred GBS328polypeptides for use with the invention comprise an amino acid sequence:(a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ IDNO: 226; and/or (b) comprising a fragment of at least ‘n’ consecutiveamino acids of SEQ ID NO: 226, wherein ‘n’ is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250or more). These GBS328 proteins include variants of SEQ ID NO: 226.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 20 226.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 226 while retaining at least oneepitope of SEQ ID NO: 226. Other fragments omit one or more proteindomains.

GBS84

The original ‘GBS84’ (SAG0907) sequence was annotated in reference 147as ‘a putative lipoprotein’ (see GI: 22533929). For reference purposes,the amino acid sequence of full length GBS84 as found in the 2603 strainis given as SEQ ID NO: 227 herein. Preferred GBS84 polypeptides for usewith the invention comprise an amino acid sequence: (a) having 60% ormore identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 227; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 227, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). TheseGBS84 proteins include variants of SEQ ID NO: 227. Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 227. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 227 while retaining at least one epitope of SEQID NO: 227. Other fragments omit one or more protein domains.

Combinations with GBS Saccharides

GBS59 polypeptides may be combined with one or more GBS capsularsaccharide(s), which will typically be conjugated to carrier protein(s).Thus the invention provides an immunogenic composition comprising acombination of:

-   -   (1) a GBS59 polypeptide as discussed above; and    -   (2) one or more GBS capsular saccharides.

A saccharide used in component (2) of this combination is ideallypresent as a conjugate comprising a saccharide moiety and a carrierprotein moiety. The carrier moiety in the conjugate may be a singleGBS59 polypeptide, a hybrid GBS59 polypeptide, a non-GBS59 GBSpolypeptide, or a non-GBS polypeptide.

The saccharide is from the capsular saccharide of GBS. The saccharidemay be a polysaccharide having the size that arises during purificationof the saccharide from bacteria, or it may be an oligosaccharideachieved by fragmentation of such a polysaccharide.

A composition may include a capsular saccharide from one or more of thefollowing streptococcal serotypes: Ia, Ib, Ia/c, II, III, IV, V, VI, VIIand VIII. A composition may include multiple serotypes e.g. 2, 3, 4, 5,6, 7, or 8 serotypes. Including a saccharide from one or more ofserotypes Ia, Ib, II, III & V is useful. The capsular saccharides ofeach of these five serotypes include: (a) a terminal N-acetyl-neuraminicacid (NeuNAc) residue (commonly referred to as sialic acid), which inall cases is linked 2→3 to a galactose residue; and (b) aN-acetyl-glucosamine residue (GlcNAc) within the trisaccharide core.

Saccharides used according to the invention may be in their native form,or may have been modified. For example, the saccharide may be shorterthan the native capsular saccharide, or may be chemically modified. Forinstance, the saccharide may be de-O-acetylated (partially or fully),de-N-acetylated (partially or fully) or N-propionated (partially orfully), etc. De-acetylation may occur before, during or afterconjugation, but preferably occurs before conjugation. Depending on theparticular saccharide, de-acetylation may or may not affectimmunogenicity. The relevance of O-acetylation on GBS saccharides invarious serotypes is discussed in ref 151, and in some embodimentsO-acetylation of sialic acid residues at positions 7, 8 and/or 9 isretained before, during and after conjugation e.g. byprotection/de-protection, by re-acetylation, etc. However, typically theGBS saccharide used in the present invention has substantially noO-acetylation of sialic acid residues at positions 7, 8 and/or 9. Theeffect of de-acetylation etc. can be assessed by routine assays. Anotherpossible modification is the removal of sialic acid residues from thesaccharide, such as side-chain terminal sialic acids [152]. Inparticular, when a serotype V capsular saccharide is used in theinvention, it may be modified by desialylation as described in ref[152]. Desialylated GBS serotype V capsular saccharide may be preparedby treating purified GBS serotype V capsular saccharide under mildlyacidic conditions (e.g. 0.1M sulphuric acid at 80° C. for 60 minutes) orby treatment with neuraminidase, as described in ref. [152]. In anotherexample, full-length polysaccharides may be depolymerised to giveshorter fragments for use with the invention e.g. by hydrolysis in mildacid, by heating, by sizing chromatography, etc. Chain length has beenreported to affect immunogenicity of GBS saccharides in rabbits [153].In particular, when a serotype II and/or III capsular saccharide is usedin the invention, it may be depolymerised as described in ref. 154. Thisdocument describes the partial depolymerization of type II and type IIIcapsular saccharides by mild deaminative cleavage to antigenic fragmentswith reducing-terminal 2,5-anhydro-D-mannose residues.

Capsular saccharides can be purified by known techniques, as describedin the references herein such as ref. 155. A typical process involvesbase extraction, centrifugation, filtration, RNase/DNase treatment,protease treatment, concentration, size exclusion chromatography,ultrafiltration, anion exchange chromatography, and furtherultrafiltration. As an alternative, the purification process describedin ref 156 can be used. This process involves base extraction,ethanol/CaCl₂ treatment, CTAB precipitation, and re-solubilisation.

The invention is not limited to saccharides purified from naturalsources, however, and the saccharides may be obtained by other methods,such as total or partial synthesis. Saccharides will typically beconjugated to a carrier protein. In general, covalent conjugation ofsaccharides to carriers enhances the immunogenicity of saccharides as itconverts them from T-independent antigens to T-dependent antigens, thusallowing priming for immunological memory.

Conjugation of GBS saccharides has been widely reported e.g. see refs.157 to 164. The typical prior art process for GBS saccharide conjugationinvolves reductive amination of a purified saccharide to a carrierprotein such as tetanus toxoid (TT) or CRM197 [158]. The reductiveamination involves an amine group on the side chain of an amino acid inthe carrier and an aldehyde group in the saccharide. As GBS capsularsaccharides do not include an aldehyde group in their natural form thenthis is typically generated before conjugation by oxidation (e.g.periodate oxidation) of a portion of the saccharide's sialic acidresidues [158, 165]. Conjugate vaccines prepared in this manner havebeen shown to be safe and immunogenic in humans for each of GBSserotypes Ia, Ib, II, III, and V [166].

Preferred carrier proteins are bacterial toxins, such as diphtheria ortetanus toxins, or toxoids or mutants thereof. These are commonly usedin conjugate vaccines. A carrier protein in a conjugate may or may notbe one of the GBS59 antigens of (1). If it is not a GBS59 antigen it mayinstead be a different GBS antigen. In some embodiments, though, thecarrier is not a GBS antigen, and may be e.g. a bacterial toxin ortoxoid.

Typical carrier proteins are diphtheria or tetanus toxoids or mutantsthereof. Fragments of toxins or toxoids can also be used e.g. fragment Cof tetanus toxoid [167]. The CRM197 mutant of diphtheria toxin [168-170]is a particularly useful with the invention. Other suitable carrierproteins include N. meningitidis outer membrane protein complex [171],synthetic peptides [172,173], heat shock proteins [174,175], pertussisproteins [176,177], cytokines [178], lymphokines [188], hormones [188],growth factors, artificial proteins comprising multiple human CD4⁺ Tcell epitopes from various pathogen-derived antigens [179] such as N19[180], protein D from H. influenzae [181-183], iron-uptake proteins[184], toxin A or B from C. difficile [185], recombinant P. aeruginosaexoprotein A (rEPA) [186], etc.

Where a composition includes more than one conjugate, each conjugate mayuse the same carrier protein or a different carrier protein.

In some embodiments, a single conjugate may carry saccharides frommultiple serotypes [187]. Usually, however, each conjugate will includesaccharide from a single serotype.

Conjugates may have excess carrier (w/w) or excess saccharide (w/w). Insome embodiments, a conjugate may include equal weights of each. Forexample, conjugates with a saccharide:protein ratio (w/w) of between 1:5and 5:1 may be used, in particular ratios between 1:5 and 2:1.

The carrier molecule may be covalently conjugated to the carrierdirectly or via a linker. Direct linkages to the protein may be achievedby, for instance, reductive amination between the saccharide and thecarrier, as described in, for example, references 188 and 189. Thesaccharide may first need to be activated e.g. by oxidation. Linkagesvia a linker group may be made using any known procedure, for example,the procedures described in references 190 and 191. A preferred type oflinkage is an adipic acid linker, which may be formed by coupling a free—NH₂ group (e.g. introduced to a glucan by amination) with adipic acid(using, for example, diimide activation), and then coupling a protein tothe resulting saccharide-adipic acid intermediate [192,193]. Anotherpreferred type of linkage is a carbonyl linker, which may be formed byreaction of a free hydroxyl group of a saccharide CDI [194, 195]followed by reaction with a protein to form a carbamate linkage. Otherlinkers include β-propionamido [196], nitrophenyl-ethylamine [197],haloacyl halides [198], glycosidic linkages [199], 6-aminocaproic acid[200], ADH [201], C₄ to C₁₂ moieties [202], etc. Carbodiimidecondensation can also be used [203].

Combinations with Non-GBS Antigens

The GBS59 clade combinations may be used in combination with non-GBSantigens. Thus the invention provides an immunogenic compositioncomprising a combination of:

-   -   (1) a combination of at least two GBS59 clades as discussed        above, as a mixture or hybrid; and    -   (2) one or more antigen(s) selected from the group consisting        of: diphtheria toxoid; tetanus toxoid; one or more pertussis        antigens; hepatitis B virus surface antigen; an inactivated        poliovirus antigen; a conjugate of the capsular saccharide        antigen from serogroup C of Neisseria meningitidis; a conjugate        of the capsular saccharide antigen from serogroup Y of Neisseria        meningitidis; a conjugate of the capsular saccharide antigen        from serogroup W135 of Neisseria meningitidis; a conjugate of        the capsular saccharide antigen from serogroup A of Neisseria        meningitides; one or more influenza antigens; and one or more        human papillomavirus antigens.

Diphtheria toxoid can be obtained by treating (e.g. using formaldehyde)diphtheria toxin from Corynebacterium diphtherias. Diphtheria toxoidsare disclosed in more detail in, for example, chapter 13 of reference204.

Tetanus toxoid can be obtained by treating (e.g. using formaldehyde)tetanus toxin from Clostridium tetani. Tetanus toxoids are disclosed inmore detail in chapter 27 of reference 204.

Pertussis antigens in vaccines are either cellular (whole cell, Pw) oracellular (Pa). The invention can use either sort of pertussis antigen.Preparation of cellular pertussis antigens is well documented (e.g. seechapter 21 of reference 204) e.g. it may be obtained by heatinactivation of phase I culture of B. pertussis. Acellular pertussisantigen(s) comprise specific purified B. pertussis antigens, eitherpurified from the native bacterium or purified after expression in arecombinant host. It is usual to use more than one acellular antigen,and so a composition may include one, two or three of the followingwell-known and well-characterized B. pertussis antigens: (1) detoxifiedpertussis toxin (pertussis toxoid, or ‘PT’); (2) filamentoushemagglutinin (‘FHA’); (3) pertactin (also known as the ‘69 kiloDaltonouter membrane protein’). FHA and pertactin may be treated withformaldehyde prior to use according to the invention. PT may bedetoxified by treatment with formaldehyde and/or glutaraldehyde but, asan alternative to this chemical detoxification procedure, it may be amutant PT in which enzymatic activity has been reduced by mutagenesis[205]. Further acellular pertussis antigens that can be used includefimbriae (e.g. agglutinogens 2 and 3).

Hepatitis B virus surface antigen (HBsAg) is the major component of thecapsid of hepatitis B virus. It is conveniently produced by recombinantexpression in a yeast, such as a Saccharomyces cerevisiae.

Inactivated poliovirus (IPV) antigens are prepared from viruses grown oncell culture and then inactivated (e.g. using formaldehyde). Becausepoliomyelitis can be caused by one of three types of poliovirus, asexplained in chapter 24 of reference 204, a composition may includethree poliovirus antigens: poliovirus Type 1 (e.g. Mahoney strain),poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g.Saukett strain).

When a composition includes one of diphtheria toxoid, tetanus toxoid oran acellular pertussis antigen in component (2) then it will usuallyinclude all three of them i.e. component (2) will include a D-T-Pacombination.

When a composition includes one of diphtheria toxoid, tetanus toxoid ora cellular pertussis antigen in component (2) then it will usuallyinclude all three of them i.e. component (2) will include a D-T-Pwcombination.

Human papillomavirus antigens include L1 capsid proteins, which canassemble to form structures known as virus-like particles (VLPs). TheVLPs can be produced by recombinant expression of L1 in yeast cells(e.g. in S. cerevisiae) or in insect cells (e.g. in Spodoptera cells,such as S. frugiperda, or in Drosophila cells). For yeast cells, plasmidvectors can carry the L1 gene(s); for insect cells, baculovirus vectorscan carry the L1 gene(s). More preferably, the composition includes L1VLPs from both HPV-16 and HPV-18 strains. This bivalent combination hasbeen shown to be highly effective [206]. In addition to HPV-16 andHPV-18 strains, it is also possible to include L1 VLPs from HPV-6 andHPV-11 strains to give a tetravalent combination.

Influenza antigens may be in the form of currently an influenza virusvaccine. Various forms of influenza virus vaccine are currentlyavailable (e.g. see chapters 17 & 18 of reference [204]). Vaccines aregenerally based either on live virus, inactivated virus, recombinanthemagglutinin or virosomes. Inactivated vaccines may be based on wholevirions, split virions, or on purified surface antigens. The antigen invaccines of the invention may take the form of a live virus or, morepreferably, an inactivated virus. The vaccine can be, for instance, atrivalent vaccine (e.g. including hemagglutinin from a A/H1N1 strain, aA/H3N2 strain and a B strain). In other embodiments the vaccine is amonovalent vaccine (e.g. including hemagglutinin from a A/H1N1 strain ora A/H5N1 strain). The vaccine can be adjuvanted (e.g. with anoil-in-water emulsion) or unadjuvanted.

Antibodies

Antibodies against GBS antigens can be used for passive immunisation[207]. Thus the invention provides a combination of antibodies forsimultaneous, separate or sequential administration, wherein thecombination includes at least two of: (a) an antibody which recognises afirst amino acid sequence as defined above; (b) an antibody whichrecognises a second amino acid sequence as defined above; and/or (c) anantibody which recognises a third amino acid sequence as defined above.

The invention also provides the use of such antibody combinations intherapy. The invention also provides the use of such antibodycombinations in the manufacture of a medicament. The invention alsoprovides a method for treating a mammal comprising the step ofadministering to the mammal an effective amount of such a combination.As described above for immunogenic compositions, these methods and usesallow a mammal to be protected against GBS infection.

Monoclonal antibodies that can be used in conjunction with the inventioninclude 17C4/A3 and 4H11/B7, which bind the surface-exposed fragment ofD3 (515 clade) at amino acids 411-436 (SEQ ID NO: 270). The heavy chainof 4H11/B7 comprises the amino acids provided in SEQ ID NO: 263 and isencoded by a nucleic acid molecule comprising SEQ ID NO: 262. The lightchain of 4H11/B7 comprises the amino acids provided in SEQ ID NO: 265and is encoded by a nucleic acid molecule comprising SEQ ID NO: 264. Theheavy chain of 17C4/A3 comprises the amino acids provided in SEQ ID NO:267 and is encoded by a nucleic acid molecule comprising SEQ ID NO: 266.The light chain of 17C4/A3 comprises the amino acids provided in SEQ IDNO: 269 and is encoded by a nucleic acid molecule comprising SEQ ID NO:268.

The present invention provides antibodies with significant sequenceidentity to 17C4/A3 and 4H11/B7 and nucleic acid molecules encoding suchantibodies. For example, the present invention includes antibodieshaving a heavy chain with greater than 60, 70, 80, 90, 95, 96, 97, 98 or99% sequence identity to the heavy chain of 4H11/B7 (SEQ ID NO: 263)and/or having a light chain with greater than 60, 70, 80, 90, 95, 96,97, 98 or 99% sequence identity to the light chain of 4H11/B7 (SEQ IDNO: 265). The present invention also includes antibodies having a heavychain with greater than 60, 70, 80, 90, 95, 96, 97, 98 or 99% sequenceidentity to the heavy chain of 17C4/A3 (SEQ ID NO: 267) and/or having alight chain with greater than 60, 70, 80, 90, 95, 96, 97, 98 or 99%sequence identity with the light chain of 17C4/A3 (SEQ ID NO: 269). Thepresent invention also includes nucleic acid molecules with greater than60, 70, 80, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:262, 264, 266, or 268.

These antibody sequences are full length sequences. The skilled personcould use the variable domains or specifically the CDR regions of theseantibodies to generate alternative antibodies such as humanisedantibodies. The invention encompasses such antibodies.

The term “antibody” includes intact immunoglobulin molecules, as well asfragments thereof which are capable of binding an antigen. These includehybrid (chimeric) antibody molecules [208, 209]; F(ab′)2 and F(ab)fragments and Fv molecules; non-covalent heterodimers [210, 211];single-chain Fv molecules (sFv) [212]; dimeric and trimeric antibodyfragment constructs; minibodies [213, 214]; humanized antibody molecules[215-217]; and any functional fragments obtained from such molecules, aswell as antibodies obtained through non-conventional processes such asphage display. Preferably, the antibodies are monoclonal antibodies.Methods of obtaining monoclonal antibodies are well known in the art.Humanised or fully-human antibodies are preferred.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references218-225, etc.

“GI” numbering is used above. A GI number, or “GenInfo Identifier”, is aseries of digits assigned consecutively to each sequence recordprocessed by NCBI when sequences are added to its databases. The GInumber bears no resemblance to the accession number of the sequencerecord. When a sequence is updated (e.g. for correction, or to add moreannotation or information) then it receives a new GI number. Thus thesequence associated with a given GI number is never changed.

Where the invention concerns an “epitope”, this epitope may be a B-cellepitope and/or a T-cell epitope. Such epitopes can be identifiedempirically (e.g. using PEPSCAN [226,227] or similar methods), or theycan be predicted (e.g. using the Jameson-Wolf antigenic index [228],matrix-based approaches [229], MAPITOPE [230], TEPITOPE [231,232],neural networks [233] OptiMer & EpiMer [234, 235]ADEPT [236]Tsites[237]hydrophilicity [238]antigenic index [239] or the methods disclosedin references 240-244, etc.). Epitopes are the parts of an antigen thatare recognised by and bind to the antigen binding sites of antibodies orT-cell receptors, and they may also be referred to as “antigenicdeterminants”.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Antibodies will generally be specific for their target. Thus they willhave a higher affinity for the target than for an irrelevant controlprotein, such as bovine serum albumin.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.245. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. 246.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, pairwise alignment between S. pneumoniae RrgB (SEQ ID NO:271)and S. agalactiae BP-2a 515 variant (SEQ ID NO:2). FIG. 1B, Model ofBP-2a 515 variant.

FIGS. 2A-2C: Identification of internal isopeptide bonds by MALDI TOFmass 5 spectrometry. The recombinant protein BP-2a 515 variant was runon a 4-12% acrylamide SDS-PAGE. The protein was “in gel” digested withLys-C. The peptides produced by the digestion were either directlyanalyzed by MALDI TOF mass spectrometry (upper panel) or were modifiedwith O-methylisourea prior the analysis (lower panel). FIG. 2A, Signalfor isopeptide bonded peptides in domain D4 (A).

FIG. 2B, Signal for isopeptide bonded peptides in domain D2; FIG. 2C,Signal for isopeptide bonded peptides in domain D3 were observed. (♦)Trypsin autodigestion product. (*) Peak not identified

FIG. 3A, SDS-PAGE of purified recombinant BP-2a 515 variant with (WT)and without isopeptide bonds (ΔIB) FIG. 3B, Opsonophagocytosis assay ofmice antisera raised against BP-2a 515 variant with (WT) and withoutisopeptide bonds (ΔIB)

FIG. 4A, schematic representation of four domains of BP-2a-515 variant.FIG. 4B, FACS analysis on 515 GBS strain with mouse sera raised againsteach BP-2a domain. FIG. 4C, Opsonophagocytosis assay with mouse seraraised against each domain of BP-2a-515 variant.

FIG. 5A, Superimposition of variants 515 and H36B. The best model forH36B variant (SAI_1511) after loop refinement reported a validationscore of 165.3 compared with the expected high score value of 215 andlow score value of 96.8. FIG. 5B, schematic representation of domains ofBP-2a H36B variant

FIG. 6A(i), D3 plus helices. FIG. 6A(ii) and FIG. 6A(iii), multiplealignment of amino acid sequences corresponding to domains D3 plus 2helices of domains D4 harbouring to allelic variant of BP-2a(D3and2H_515, SEQ ID NO:55; D3and2H_CJB111, SEQ ID NO:59); D3and2H_DK21,SEQ ID NO:71; D3and2H_O9O, SEQ ID NO:67; D3and2H_H36B, SEQ ID NO:63; andD3and2H_2603, SEQ ID NO:51). FIG. 6B, schematic representation of fusionproteins. FIG. 6C, SDS-PAGE of purified recombinant fusion proteinsdetected by COOMASSIE® staining. FIG. 6D, FACS analysis on GBS strainsexpressing different BP-2a variants with mouse antisera sera raisedagainst fusion protein 6×D3. FIG. 6E, Opsonophagocytosis assay withmouse antisera raised against fusion proteins 6×D3 and 4×D3H.

FIGS. 7A-7D(iii): Crystal structure of BP-2a-515 solved and refined at1.75 Å resolution via molecular replacement. Data collection andrefinement statistics are shown in Table 2.

The crystal asymmetric unit contains a dimer of two independent chains ,each made up of three distinct domains: D2 (residues 190-332), D3(residues 333-455) and D4 (residues 456-641). FIG. 7A, residues 192-640.FIG. 7B, residues 190-641). FIG. 7C(i), IP1. FIG. 7C(ii), IP2. FIG.7C(iii), IP3. FIG. 7D(i), mass spectrometry spectra (NTETKPQVDKNFADK,amino acids 21-34 of SEQ ID NO:57; ITYSATLNGSAVVEVLETNDVK, amino acids152-173 of SEQ ID NO:56). FIG. 7D(ii), mass spectrometry spectra(ITVNKTWAVDGNEVNK, amino acids 20-35 of SEQ ID NO:38). FIG. 7D(iii),mass spectrometry spectra (FVKTNK, amino acids 130-135 of SEQ ID NO:14;DAQQVINKK, amino acids 159-169 of SEQ ID NO:15).

MODES FOR CARRYING OUT THE INVENTION

Streptococcus agalactiae (Group B Streptococcus [GBS]) is the mostcommon cause of sepsis and meningitis in neonates and is also theprimary colonizer of the anogenital mucosa of healthy women. Recently,three pilus types have been discovered in GBS as important virulencefactors. The genes involved in pilus assembly are clustered incharacteristic genomic loci (named PI-1, PI-2a and PI-2b), each encodingthree proteins containing a LPXTG (SEQ ID NO:272) motif representing thestructural components of the pilus, and two sortase enzymes catalyzingprotein polymerization. Each of the three pilus types carries twoprotective antigens. Among these, the backbone protein of pilus type 2a(BP-2a), showed the highest degree of gene variability and was able tosignificantly mediate opsonophagocytic activity and to confer protectionin mice only against strains expressing the homologous allele. In orderto map immunodominant and protective epitopes of the allelic variants ofBP-2a, we performed a structural characterization of the protein bycomparative homology modeling and on the basis of this structuralinformation, we generated deletion mutants of the main variantscorresponding to the four IgG-like fold domains identified. In vitro andin vivo studies showed that only the C-terminal portion of the proteinwas highly surface-exposed and able to elicit opsonophagocyticantibodies conferring protection in mice. In particular, domain D3appeared to be the most important for the protective immunity of themain four allelic variants analyzed. Finally, we showed that a broadprotective vaccine against GBS infection can be generated with a fusionprotein containing D3 domains from different BP-2a variants.

Materials and Methods

Comparative Homology Modelling

All molecular simulations were performed using Discovery Studio 2.5software from Accelrys, USA. The amino acid sequences of the BP-2a (515variant, TIGR annotation SAL_1486 and H36B variant, TIGR annotationSAI_1511) were used to search against the Protein Data Bank (PDB) withthe BLAST program tool [247]. The best template structure for bothprotein sequences for homology modeling resulted to be the crystalportion (residues 187 to 627) of RrgB (PDB code: 650), the backboneprotein of S. pneumoniae pilus, that was obtained from the PDB database.Pairwise sequence alignment between SAL_1486 and RrgB and betweenSAI_1115 and RrgB were done using multiple sequence alignment tool in DS2.5 followed by manual modifications to improve the alignment quality.The models were generated with MODELER [248] from protein modelingmodule of DS 2.5, performing both homology modeling and loop refiningfor the protein. Ten models have been generated and the model whichshared the least RMS deviation with respect to trace (Ca atoms) of thecrystal structure of the template, was selected for further refinementsand validations. The quality of the refined structure obtained forSAL_1486 was checked with verify Profile-3D module in DS 2.5, and itsstereochemical quality was examined by Ramachandran plot using DS 2.5.In order to optimize particular loop regions in the generated structurefor SAI_1511, the loop refinement module, based on CHARMm and Loopermolecular mechanics, of DS 2.5 was used. Finally, the model structuresgenerated have been superimposed using Align Structures module of DS2.5.

Protein Crystallization

Crystallization trials were set up in 96-well microbatch plates(Greiner) using the Orxy 8.0 crystallization robot (DouglasInstruments). Crystals of BP-2a-515 grew after one to two weeks at 20°C. in a 0.5 μl drop consisting of 0.3 μl protein (180 mg/ml) in 10 mMHEPES pH 7.0 and 0.2 μl crystallization solution (25% (w/v) PEG 4000,0.1 M HEPES pH 7.0 and 90 mM potassium sodium tartrate tetrahydrate),layered with silicon oil and paraffin, mixed at a ratio of 1:1. Crystalswere cryoprotected in the crystallization solution containing increasedprecipitant concentration (40% (w/v) PEG4000). Crystals belong to theorthorhombic P2₁2₁2₁ space group with two BP-2a 515 chains present inthe asymmetric unit, and an estimated solvent content of 53%.

Structure Solution and Refinement

Diffraction data from a single crystal were collected at a resolution of1.75 Å at the European Synchrotron Radiation Facility (Grenoble, France;beam line ID23-1). Data were processed using isofilm (32) and SCALA (33)available from the CCP4 Program Suite (34). The crystal structure ofBP-2a-515 was solved by molecular replacement using the program Molrep(35) and the structure of the pilus backbone protein (RrgB) from S.pneumoniae (15) (PDB 2X9W), as a search model. The initial Molrep outputmodel was extended using ARP/wARP (36). The structure was refined usingREFMAC 5 (37) and modeled to electron density maps using Coot (38). Thelatter stages of refinement included the translational-libration-screw(TLS) option (39). During model building and refinement, it becameapparent that the protein had been cleaved at the N-terminus, lackingapproximately 190 residues, as previously observed for RrgB (15). Thefinal model displays optimal stereochemical geometric parameters with99.1% of residues in the most favorable regions of the Ramachandranplot, with no outliers, according to validation carried out usingMolProbity (molprobity.biochem.duke.edu/) (40, 41). Atomic coordinatesand structure factors for residues 190-640 of BP-2a-515 have beendeposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics, Rutgers University, New Brunswick, N.J.(www.rcsb.org) under accession code 2XTL (Reference to add PDB: Berman,H. M., et al., The Protein Data Bank. Nucleic Acids Res, 2000. 28(1): p.235-42.).

Bacterial Strains and Growth Conditions

The GBS strains used in this work were 2603 V/R (serotype V), 515 (Ia),CJB111 (V), H36B (serotype Ib), 5401 (II) and 3050 (II). Bacteria weregrown at 37° C. in Todd Hewitt Broth (THB; Difco Laboratories) or intrypticase soy agar supplemented with 5% sheep blood.

Cloning, Expression, Purification of Recombinant Proteins and Antisera.

GBS strains 515 and H36B were used as source of DNA for cloning thesequences coding for the single domains (D1, D2, D3 and D4) of the BP-2a515 and H36B allelic variants. Genomic DNA was isolated by a standardprotocol for gram-positive bacteria using a NucleoSpin Tissue kit(Macherey-Nagel) according to the manufacturer's instructions. Genescorresponding to each domain were first cloned into pENTR™/TEWD-TOPOvector (Invitrogen) and then sub-cloned into pET54 DEST vector(N-terminal 6× HIS tag) or pET59 DEST (N-terminal 6× His-TRX tag)(Novagen) using the GATEWAY cloning system (Invitrogen), with theexception of D3 domain of SAI_1511, which was cloned in the pSpeedETvector by PIPE cloning method [249]. The oligos used are listed in Table1.The resulting constructs were checked for sequencing and thentransformed into E. coli BL21(DE3) (Novagen) for the expression as 6His-or TRX-tagged fusion proteins.

The full length recombinant BP-2a proteins, corresponding to 515, CJB111and 2603 allelic variants (TIGR annotation SAL1486, SAM1372 and SAG1407,respectively), were produced as previously reported [2], while the fulllength H36B variant (TIGR annotation SAI_1511) was cloned in pET24b+(Novagen) using strain H36B as source of DNA. Primers were designed toamplify the coding regions without the signal peptide and the 3′terminal sequence starting from the LPXTG (SEQ ID NO:272) motif.

The genes coding for the BP-2a fusion proteins, 6XD3 and 4XD3-H, weresynthetically constructed from GENEART. The 6XD3 gene was then clonedinto pET15 vector adapted in house using PIPE cloning in E. coli HK100strain. The 4XD3-H gene was sub-cloned using NdeI and XhoI restrictionenzymes into the expression vector pColdI (N-terminal 6× HIS-tag,Takara). The final constructs were sequenced and transformed inBL21(DE3) (Novagen).

For the recombinant protein expression, the cultures were maintained at25° C. for 5 h after induction with 1 mM IPTG for the pET clones or with0.2% arabinose for the SpeedET clones. All recombinant proteins werepurified by affinity chromatography and gel filtration. Briefly, cellswere harvested by centrifugation and lysed in “lysis buffer”, containing10 mM imidazole, 1 mg\ml lysozyme, 0.5 mg\ml DNAse and COMPLETEinhibitors cocktail (Roche) in PBS. The lysate was clarified bycentrifugation and applied onto His-Trap HP column (AmershamBiosciences) pre-equilibrated in PBS containing 10 mM imidazole. Proteinelution was performed using the same buffer containing 250 mM imidazole,after two wash steps using 20 mM and 50 mM imidazole buffers. The elutedproteins were then concentrated and loaded onto HiLoad 16/60 Superdex 75(Amersham Biosciences) pre-equilibrated in PBS. For the expression ofthe 4XD3-H was maintained at 37° C. until OD 600 nm reached the value of0.7 and after induction in the presence of 1 mM IPTG , the temperaturewas switched to 20° C. and the culture was maintained at thistemperature overnight. Protein concentration of the pure fractions wasestimated using BCA assay (PIERCE).

Antisera specific for each protein were produced by immunizing CD1 micewith the purified recombinant proteins as previously described [250].Protein-specific immune responses (total Ig) in pooled sera weremonitored by ELISA.

Site-Directed Mutagenesis

For the generation of the mutated form of BP-2a 515 variant, containingthe lysine residues involved in the isopeptide bonds mutated intoalanine, mutations were introduced into the wild type BP-2a 515 variantcarrying the LPXTG (SEQ ID NO:272) motif. Primers used for theamplification of the gene coding for BP-2a 515 with LPXTG (SEQ IDNO:272) motif are listed in Table 1. Site-directed mutagenesis wasperformed using the PIPE method and forward and reverse primer pairs foreach mutation were designed, as listed in Table 1. The wild type proteinand the mutated form, were cloned into SpeedET vector (N-term 6× Histag) and expressed in E. coli HK100 strain. The sequences of theresulting constructs were confirmed by DNA sequencing. Proteins werepurified by affinity chromatography and gel filtration as describedabove.

Flow Cytometry

Mouse sera raised against purified deletion mutant of the 515 and H36Bvariants were analyzed on whole bacteria by flow cytometry to evaluatethe surface-exposure of the corresponding domains. Exponential phasebacterial cells were fixed in the presence of 0.08% (wt/vol)paraformaldehyde and incubated for 1 h at 37° C. Fixed bacteria werethen washed once with PBS, resuspended in Newborn Calf Serum (Sigma) andincubated for 20 min. at 25° C. The cells were then incubated for 1 hourat 4° C. in pre-immune or immune sera, diluted 1:200 in dilution buffer(PBS, 20% Newborn Calf Serum, 0.1% BSA). Cells were washed in PBS-01%BSA and incubated for a further 1 h at 4° C. with a 1:100 dilution ofR-Phycoerythrin conjugated F(ab)2 goat anti-mouse IgG (JacksonImmunoResearch Laboratories; Inc.). After washing, cells wereresuspended in PBS and analyzed with a FACS Calibur apparatus (BectonDickinson, Franklin Lakes, N.J.) using FlowJo Software (Tree Star,Ashland, Oreg.). Data are expressed as the difference in fluorescencebetween cells stained with immune sera versus pre-immune sera.

Immunoblotting

Group B Streptococcus strains were grown overnight in THB (DifcoLaboratories, Detroit, Mich.) to exponential phase (0D600=0.5). Bacteriawere pelleted, washed in PBS and resuspended in 50 mM Tris-HCl,containing 400 units of mutanolysin (Sigma-Aldrich). Bacterialsuspension was then incubated 2 h at 37° C. and lysed by freeze andthaw. Cellular debris were removed by centrifugation at 14 000 rpm for10 min and protein concentration measured by Bio-Rad Protein assay.Total cell proteins were separated by 4-12% NuPage Novex pre-cast gels(Invitrogen) and electroblotted onto PVDF membranes using the iBlot™ DryBlotting System (Invitrogen). After blocking in 1× x phosphate-bufferedsaline (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mm KH2PO4,pH 7.3) containing 0.05% Tween 20 and 10% skim milk for 1 h at roomtemperature, membranes were incubated for 1 h at room temperature (RT)with primary antibodies diluted 1:500. After washing three times in PBScontaining 0.05% Tween 20 (PBST), the membranes were incubated for 1 hwith horseradish peroxidase-conjugated secondary antibodies (Dako).Positive bands were visualized with the Opti-4CN Substrate Kit(Bio-Rad).

Opsonophagocytosis Assay

The opsonophagocytosis assay was performed using differentiated HL-60 asphagocytic cells and strains 515, CJB111, 3050 and 5401 as target cells.GBS strains were grown in Todd-Hewitt broth (THB) to mid-exponentialgrowth phase (A650 nm=0.3). The bacteria were harvested bycentrifugation, washed twice with cold saline solution, and finallyresuspended in HBSS buffer (Invitrogen) to a concentration of ≈1.2×10⁷CFU/ml. Promyelocytic HL-60 cells (ATCC, CCL-240) were expanded in RPMI1640 (Gibco, Invitrogen) containing 10% Fetal clone I (HyClone) at 37°C. with 5% CO₂ and differentiated into granulocyte-like cells to adensity of 4×10⁵ cells/ml by the addition of 100 mM N, Ndimethylformamide (DMF, Sigma) to the growth medium. After 4 days, cellswere harvested by centrifugation and resuspended in HBSS buffer to aconcentration of ≅4 ×10⁷ cells/ml. In brief, the reactions took place ina total volume of 125 μl containing ≈3×10⁶ differentiated HL-60,≈1.5×10⁵ CFU of GBS cells, 10% baby rabbit complement (Cedarlane), andheat-inactivated mouse antisera at 37° C. for 1 h with shaking at 600rpm. Immediately before and after 1 h of incubation, a 25-μl aliquot wasdiluted in sterile distilled water and plated onto trypticase soy agarplates with 5% sheep blood. A set of negative controls included in eachexperiment consisted of reactions containing preimmune sera, reactionswithout HL-60, and reactions with heat-inactivated complement. Theamount of opsonophagocytic killing (log kill) was determined bysubtracting the log of the number of colonies surviving the 1 h assayfrom the log of the number of CFU at the zero time point.

Mouse Active Maternal Immunization Model

A maternal immunization/neonatal pup challenge model of GBS infectionwas used to verify the protective efficacy of the produced proteins inmice, as previously described (Maione et al., 2005). Briefly, CD-1female mice (6-8 weeks old) were immunized on days 1 (in CFA), 21 and 35(IFA) with either PBS or 20 mg of recombinant protein and were then bred3 days after the last immunization. Within 48 h of birth, pups wereinjected intraperitoneally with a dose of different GBS strainscalculated to cause 90% lethality. Survival of pups was monitored for 2days after challenge. Statistical analysis was performed using Fisher'sexact test. All animal studies were performed according to guidelines ofthe Istituto Superiore di Sanita (Italy).

Results

We have previously shown that the Backbone Protein of pilus 2a (BP-2a)in Group B Streptococcus is able to confer protection in an activematernal/pup challenge model in mouse [1]. However, the existence ofseven highly variable allelic variants and the demonstration that eachvariant confers protection only against the homologous strain restrictsthe possibility of using this antigen alone or in combination with otherantigens in a broad-spectrum vaccine against GBS [2]. Nevertheless,BP-2a is an antigen of interest since it is able to promote high levelsof opsonic killing of GBS when tested in an opsonophagocytosis assay inthe presence of specific antibodies. We used this specific feature inorder to explore the protective capability of this antigen and identifythe immunodominant epitopes of BP-2a.

Comparative Homology Modeling of the BP-2a 515 Variant

In order to design appropriate deletion mutants of BP-2a, we performed astructural characterization of the protein, first focusing on the 515variant (TIGR annotation SAL_1486), by comparative homology modeling.The PDB was searched for a protein sharing significant sequence identitywith BP-2a. The best template structure found was PBD code 650corresponding to the RrgB pilus protein of S. pneumoniae. The RrgBcrystal comprises the region from residues 187 to 647 and it is arrangedin three immunoglobulin-like domains, each one carrying a stabilizingisopeptide bond. The first two domains (D2 and D3) are closely packed toform a compact structure from which two anti-parallel helixes and thethird domain (D4) protrude. The PDB 650 crystal does not include thefirst domain (D1) of the RrgB protein.

The amino acid sequences of BP-2a 515 variant and RrgB were aligned andreported to share 43% sequence identity and 61% sequence similarity.Sequence comparison revealed that the pilin motif YPK, the E-Boxcassette, the LPXTG (SEQ ID NO:272) motif and all the residues involvedin isopeptide bonds are well conserved (FIG. 1A). The pairwise alignmentwas further manually optimized to cope with the secondary structures inorder to refine the homology modeling procedure input. The quality ofthe model was assessed by calculating the compatibility score withProfile-3D module (Discovery Studio 2.5 Software Inc., San Diego,Calif.). The template reported a score of 168.6 compared with theexpected high score value of 201.7 and low score value of 90.7. The bestmodel for BP-2a-515 reported a validation score as computed byProfile-3D of 154, while the minimum and the maximum possible scores forthis model are 92.2379 and 204.973, respectively. Given that even thetemplate crystal structure does not reach the expected score for thecorrectly folded protein, the model obtained a validation scorecomparable to the crystal one.

As shown in FIG. 1B, the model of our protein, corresponding to aminoacid residues 191 to 640, revealed three IgG-like fold domains (D2, D3and D4), each one characterized by a stabilizing isopeptide bond.Superimposing the template structure against the generated model, a RMSDas low as 1.2 Å is obtained, meaning that the sequence of BP-2a fittedwell in the template structure. Only minor differences in loops wereidentified, all due to short residue insertions or deletions. Moreover,the superimposition of crystallized isopeptide bonds shows that theregion in the vicinity of the bonds is relatively conserved. Inparticular, there is a glutamic acid or an aspartatic acid whichcatalyze bonds, completely immersed in a surrounding hydrophobic cavity.In the BP-2a 515 variant, the residues involved in isopeptide bonds are:Lys199-Asn325 in the D2 domain with Asp 247 as the catalyzing andstabilizing residue; Lys355-Asn437 in the D3 domain with Glu416; andLys463-Asn636 in the D4 domain with Glu589. Based on secondary structureand fold prediction of the N-terminal portion of the BP-2a protein(residues 1 to 190), we hypothesise that this portion has the sameIgG-like fold of D2, D3 and D4 (data not shown).

Mass Spectrometry Analysis Confirms the Presence of Three InternalIsopeptide Bonds

Recombinant full length SAL_1486 was purified and used to confirm thepresence of the isopeptide bonds hypothesized from the modeling study.The method selected for their identification was based on the totaldigestion of the diverse constructs with Lys-C and analysis of thedigestion products by mass spectrometry. In order to easily sort thebond peptides, the digestion products were derivatized withO-methylisourea that modifies the C-terminal lysine in homoarginine witha mass increase of 42 Da for each modified C-terminal extremity.Isopeptide bonded peptides are those presenting a shift of mass of 42 D(partial derivatization) and 84 Da (complete derivatization). Theprotein was resistant to “in solution” enymatic digestions (data notshown). The approach that allowed the larger peptide coverage wasobtained from “in gel” digestion of the polypeptide run on SDS-PAGE.FIG. 2 reports the mass spectrometry spectra obtained from the fulllength recombinant SAL_1486 allowing the confirmation of thehypothesized isopeptides.

An isopeptide bond involving amino acids carried by the D4 domain of theprotein was evidenced by the molecular ion of m/z 1762.05 Da thatcorresponds to the molecular mass of the peptide 461_(FVKTNK) ⁴⁶⁶ (aminoacids 130-135 of SEQ ID NO:14) linked by an isopeptide bond to thepeptide ⁶³⁰DAQQVINKK⁶³⁸ (amino acids 159-169 of SEQ ID NO:15) (expectedmolecular mass 1761.90 Da) (FIG. 2A, upper panel). The guanidinationreaction induced a shift of 42 and 84 Da of the signal that correspondsto the single and double C-terminal peptide derivatization,respectively, confirming the covalent linkage of the two peptides. Inthe same way, isopeptide bonds in domains D2 and D3 were assigned fromthe ions of m/z 2145.18 and 4040.85 that correspond to the molecularmass of peptide ⁵³ITVNKTWAVDGNEVNK⁶⁸ (amino acids 20-35 of SEQ ID NO:38)linked to peptide ¹³⁹NNK¹⁴¹ (expected molecular mass 2145.13 Da), and ofpeptide ¹⁸⁵ITYSATLNGSAVVEVLETNDVK²⁰⁶ (amino acids 152-173 of SEQ IDNO:56) linked by an isopeptide bond to the peptide ⁶⁸NTETKPQVDKNFADK⁸²(amino acids 21-34 of SEQ ID NO:57) (expected molecular mass 4040.07Da), respectively (FIGS. 2B and 2C). The guanidination reactionconfirmed the covalent linkage of the peptides by the double shift ofmass of 42 and 84 Da. It was noteworthy that no isopeptide bond wasidentified in the N-terminal part corresponding to domain D1 of thefull-length recombinant protein.

In order to confirm that the lysines involved in the isopeptide bonds ofdomains D2, D3 and D4 corresponded exactly to K199, K355 and K463predicted by structural model, we generated a recombinant form of theprotein by site-directed mutagenesis in which these lysine residues weremutated into alanine residues (K199A/K355A/K463A). The same protocol ofenzymatic digestion and mass spectrometry analysis was applied, and noneof the signals, corresponding with isopeptide linked peptides andreported above, were identified (data not shown).

X-ray Crystal Structure of BP-2a-515 Pilus Subunit

With the aim to identify the domain(s) carrying the protective epitopes,the crystal structure of BP-2a-515 was solved and refined at 1.75 Åresolution via molecular replacement. Data collection and refinementstatistics are shown in Table 2. The crystal asymmetric unit wasconfirmed to contain a dimer of two independent chains (A: residues192-640 and B: residues 190-641), each made up of three distinctdomains: D2 (residues 190-332), D3 (residues 333-455) and D4 (residues456-641) (FIG. 7). The observed BP-2a-515 dimer does not displayextensive intermolecular interactions at the association interface,therefore the dimer is not expected to occur in solution and it is alikely consequence of crystal packing, as indicated by the ProteinInterfaces, Surfaces and Assemblies (PISA) Service [251] at the EuropeanBioinformatics Institute (www.ebi.ac.uk/msd-srv/prot_int/pistart.html).

Although crystallization was carried out using the full-length protein,approximately 190 amino acids from the N-terminus (D1 domain) wereabsent in the crystal, suggesting that they are cleaved off prior tocrystallization. A similar behavior was reported for the pneumococcalRrgB pilus protein whose structure was recently solved at 1,6 Åresolution [252] and is highly homologous to the structure of BP-2a-515.

Potassium-sodium tartrate present in the crystallization solution wasrelevant for optimizing crystal growth and improving diffractionresolution. In fact, three potassium cations are bound at strategic andstabilizing positions in the structure. Two (identically-coordinated)potassium cations are bound to the D2 domain in both chains andstabilize a flexible linker, connecting the D3 and D4 domains, viacontributing residues from domains D2, D3, and a water molecule (FIG.7). The third potassium cation stabilizes a flexible loop in the D4domain from chain A.

The organization of the three domains was confirmed to show a modifiedIgG fold [252], a structural feature already observed for RrgB of S.pneumoniae. Indeed, superimposition of the C-alpha atoms of BP-2a-515chain B and RrgB using the pairwise structural alignment C-alpha matchprogram (bioinfo3d.cs.tau.ac.il/c_alpha_match/), yields a r.m.s.d valueof 1.37 Å over 280/452 residues. The major structural differencesbetween the two proteins regard the spatial location of the D3 domain,the movement of two α-helices in the D4 domain that are connected to theβ-sandwich by two β-strands not present in RrgB, and flexible regions.

Similarly to RrgB, each domain is characterized by a stabilizing,covalent intramolecular isopeptide bond, formed between the ε-aminogroup of lysine side chains and the δ-carboxyamide group of asparagine.The three isopeptide bonds occur between Lys199 and Asn325 (D2 domain),Lys437 and Asn355 (D3 domain), and Lys463 and Asn636 (D4 domain), andstabilize the secondary structural elements of their respective domains.Due to the conformational movement of D3 in comparison with RrgB, thelatter isopeptide bond is the only one that does not match the spatiallocation of the equivalent bond in RrgB. The surrounding area aroundthese bonds is largely hydrophobic, comprising several aromaticresidues, in agreement with observations made for the isopeptide bondsin several pilus proteins.

Each of the four domains D1, D2, D3 and D4 appear to fold independently.This was demonstrated by expressing and purifying each domain from E.coli, as independent constructs whose N and C termini were selected onthe basis of the domain boundaries defined in the crystal structure ofBP-2a-515. All four domains were expressed in soluble form in E. coli,and Mass Spectrometry analysis of tryptic digests of D2, D3 and D4revealed that the domains carried the same isopeptide bonds found in thefull-length protein data not shown). This suggested that the overallstructural organization of the independently expressed domains wassufficiently preserved to bring the lysine and asparagine residues at asuitable reaction distance.

In conclusion, the crystal structure of the backbone subunit of PI-2a(515 allele) indicates that the protein is organized into four domains,which are shown to be independently structured and stable.

Intramolecular Isopeptide Bonds are Dispensable for Protection

It has been demonstrated that intramolecular isopeptide bonds that aredispensable for pilus assembly, contribute to structural and proteolyticstability of pili. The SD S-PAGE of the wild-type and mutated BP-2aprotein showed that the protein without isopeptide bonds had a slowerelectrophoretic mobility compared to the wild-type form. The presence ofinternal cross-links within the naive protein may make the wild-typeprotein structure more compact and more able to pass through the matrixof the gel, whereas the mutated form has a larger structure which runsto a higher molecular weight (FIG. 3A).

In order to evaluate if the presence of these internal linkages couldinfluence the protective capability of the protein BP-2a and investigateif the mutant protein is able to induce protective immunity in vivo aswell as the wild type, we tested both proteins in a mouse maternalimmunization model [250]. We immunized groups of adult female CD1 micewith the purified recombinant proteins and after three immunizations,mice were mated and the resulting offspring were challenged with a doseof GBS calculated to kill about 90% of the pups. The high levels ofprotection observed with the mutated form of the protein (Table 3)revealed that the loss of isopeptide bonds did not interfere withcapacity of the protein to confer protection in mice and to elicitopsonic antibodies (FIG. 3B).

Domain D3 is Highly Surface Exposed and Essential for Protection

Based on the information obtained from the structural model describedabove, we generated four deletion mutants of the BP-2a 515 variant,dividing the protein in four overlapping fragments corresponding to thefour IgG-like domains predicted by modeling (FIG. 4A): D1 corresponds tothe region from amino acids 30 to 162; D2 corresponds to the region fromamino acids 156 to 338; D3 corresponds to the region from amino acids332 to 499; and D4 corresponds to the region from amino acids 457 to640.

The deletion fragments were cloned, expressed in E. coli and purified asHIS- or TRX-tagged recombinant proteins, as described in Materials andMethods. Interestingly, the isopeptide bonds present into domains D2 andD3 and D4 were also identified in the single recombinant forms of thesedomains, indicating that the single domain had all the requirements forthe formation of this covalent bond (data not shown).

The four purified soluble domains were used for immunizing CD1 mice andprotein-specific immune responses (i.e., the total immunoglobulin level)were monitored by ELISA and Western Blotting. Sera raised against eachfragment were also analyzed by flow cytometry using whole bacteriastrain 515 in order to evaluate which domain was exposed on thepolymerized pilus protruding from bacterial surface. As shown in FIG.4B, domains D3 and D4 were highly exposed at a level comparable to thoseobserved with the antiserum raised against the full length protein. Aweak shift was obtained using antibodies anti-D2 suggesting it is nothighly exposed, whereas D1 was not exposed.

To investigate which of the domains were able to confer protectionagainst GBS infection, we performed an in vitro opsonophagocytosisanalysis using sera from immunized mice and an in vivo active maternalmouse immunization/neonatal pup challenge model. According to the FACSresults, only domains D3 and D4 domains were able to elicitopsonophagocytic antibodies and confer protection in mice against GBSinfection (FIG. 4C and Tables 3 and 4). In particular, domain D3 showedthe highest level of surface exposure and opsonic activity.

The selection of FACS positive and opsonic mAbs mapping in the D3 domainconfirmed that the C-terminal portion of the protein and in particularD3 is essential for protective immunity (data not shown).

Domain D3 Represents the Immunodominant Epitope of the Main AllelicVariants of BP-2a

We have observed that all the allelic variants described so far, sharinga sequence homology ranging from 48% to 98%, were protective in mousemodel, although they protected only pups challenged with strainscarrying the allelic variant used to immunize the respective mothers([2] and data not shown).

To investigate if the results obtained with the 515 allele wereconfirmed in the other variants, we applied the same approach describedabove to map the immunodominant portion in the most representativevariants (named 515, CJB111, H36B and 2603) belonging to the two majorfamilies.

In order to understand if the BP-2a variants shared the same structuralorganization, a new structural model of the H36B allele (TIGR annotationSAI_1511), was generated. This variant was chosen because it is the mostdivergent in terms of sequences identity and similarity, from the 515variant (48% of sequence identity). The RrgB pilus protein of S.pneumoniae was used as template structure (PDB code: 650). The aminoacid sequences of SAI_1511 and RrgB were aligned and reported to share38% sequence identity and 56% sequence similarity. The model ofSAI_1511, as reported in FIG. 5A, revealed the same modular structure asthe 515 variant, comprising 4 IgG-like fold domains, 3 of which containinternal Lys-Asn isopeptide with relatively conserved surroundings.Moreover, even though the main protein structure organization isconserved (RMSD=0,9 Å), in the H36B variant model structure there aretwo insertion loops which are not present in RrgB crystal structure. Thefirst one spans residues 200 to 214, while the second one spans residues402 to 410 (FIG. 5A). The function of these two additional loop regionsis still unknown.

Based on the information obtained from structural analysis, we generateddeletion mutants of the H36B variant, dividing this variant in fouroverlapping fragments, expressed in E. coli and purified as recombinantproteins. D1 corresponded to the region from amino acids 30 to 158, D2to the region from amino acids 152 to 350, D3 to the region from aminoacids 343 to 493 and D4 to the region from amino acids 487 to 658 (FIG.5B). The purified soluble domains were used to immunize CD1 mice andsera raised against each fragments were tested in in vitro and in vivoprotection assays.

As observed for the 515 variant, antisera raised against domain D3showed the highest fluorescence shift when tested in Flow CytometryAnalysis on whole bacterial cells (FIG. 5C), and was able to promoteefficient killing of bacteria when analyzed in an opsonophagocytosisassay in presence of human polymorphonuclear leukocytes (PMNs) and babyrabbit complement (FIG. 5D). In addition, domain D3 conferredsignificant levels of protection against the challenge strain in whichthe H36B variant was well expressed and exposed on the bacterial surface(Tables 5 and 6).

Domain D3 has further been confirmed as the immunodominant epitope forBP-2a in all the known allelic variants (data not shown). For example,as shown in Table 7, domain D3 from the CJB111 variant conferssignificant levels of protection against challenge with the CJB111strain. In addition, two monoclonal antibodies (17C4/A3 and 4H11/B7, SEQID NOs: 262-269) have been found to bind an epitope comprising aminoacids 411-436 (SEQ ID NO: 270) within the D3 sub-fragment from the 515clade (SEQ ID NO: 38, fragment of SEQ ID NO: 2) (data not shown).

Fusion Proteins Carrying Protective Epitopes Confer Cross ProtectionAgainst GBS Strains Expressing Different Alleles.

The mapping of the D3 domains as the immunodominant and protectiveregion of the different variants was then used to facilitate the designof chimeric proteins to test in the animal model in order to evaluatethe ability of these fusion proteins to confer broad-spectrumprotection.

Two fusion proteins were generated. The first one, fusion protein 6XD3,is composed of domain D3 of six backbone protein variants (515, CJB111,H36B, DK21, 090 and 2603) (FIGS. 6A and B). The second one, fusionprotein 4XD3Helix, is composed of domain D3 plus the two protrudinghelices of domain D4 of the four most representative variants (515,CJB111, H36B and 2603) (FIGS. 6A and B). Each fusion protein has beencloned and expressed in E. coli and purified as recombinant proteins, asreported in Material and Methods (FIG. 6C).

The purified fusion proteins were used to immunize CD1 mice and seraraised against each fusion protein were tested in in vitro and in vivoprotection assays. In order to understand if the sera were able torecognize pilus-like structures containing the different variants ofBP-2a, we performed Western Blotting and FACS analysis (data not shownand FIG. 6D). The results showed that the immune sera generated againstthe two fusion proteins are able to recognize the pilus-like structuresfrom the total extracts. Opsonophagocytosis experiments confirmed thatantisera against fusion proteins were able to mediatecomplement-dependent phagocytic killing of GBS strains expressingdifferent allelic variants of BP-2a (FIG. 6E). In addition, data fromthe in vivo protection model show that both fusion proteins were able toelicit protective immunity in mice challenged with strains expressingthe different variant of the protein and can thus be used as abroadly-protective vaccine against GBS infections (Tables 8 and 9).Finally, Table 9 shows that the protective effect of the 6XD3 fusion ismaintained if a His tag is used.

Discussion

Many bacterial pathogens, including S. agalactiae (GBS), have evolved awide range of mechanisms to escape the immune system of their hosts orto adapt to environmental variation, for instance, adopting the strategyof gene variability and/or differential gene expression. Thesestrategies play a crucial role in the capacity of pathogens to triggerdisease and also explain why it is so difficult to develop vaccinesagainst these microorganisms. Advances in sequencing technology andbioinformatics have resulted in an exponential growth of genome sequenceinformation and complete genomes of multiple isolates are now availablefor a large number of pathogens. Multigenome analysis has revealedunexpectedly high gene variation between strains of a single species,with implications for effective vaccine and drug-discovery programs.Species such as streptococci may have a relatively small genome, but thetotal number of dispensable genes in the population permits sufficientflexibility for the species to adapt to environmental challenge.

Pathogenicity islands, such as pilus islands discovered in GBS and inother Gram-positive pathogens in the recent years, belong to the classof genomic islands, which have been acquired by horizontal gene transferand are a typical example of dispensable genome. Because they promotegenetic variability, genomic islands play an important role in microbialevolution. The three pilus islands identified in GBS (named PI-1, PI-2aand PI-2b) encode high molecular weight structures whose subunits arepotential protein vaccine candidates. However, since pilin antigens arenot universally present, conserved and expressed on the bacterialsurface of a large subpopulation of GBS, only a combination of moreproteins would be suitable for a broad-spectrum vaccine.

The backbone protein of pilus 2a (BP-2a), is essential for piluspolymerization. Although BP-2a is able to confer protection in mice andto mediate opsonophagocytic killing of live GBS bacteria at a levelcomparable to killing observed with antibodies against capsularpolysaccharide antigens, it has the highest level of gene variabilityamong all pilin antigens. The existence of at least seven non-crossprotective allelic variants of BP-2a blocks the possibility to use thisantigen alone for a broad-spectrum vaccine, except by including all theidentified alleles in the vaccine.

For an immunogenic multi-variant antigen such as BP-2a, the selection ofonly a small protective portion of the protein (the highlysurface-exposed IgG-like fold domain D3 of the protein) allowed us torationally design and produce chimeric proteins by fusion of the singleimmunodominant domains from the different non-cross-reacting alleles.These chimeras acquired the capability to confer broad cross-protectionin mice against infections from GBS strains expressing all BP-2avariants. The combined approach of structural and functional analysisreported herein, together to use of tools of genetic engineering allowedus generate fusion proteins containing the immunodominant domain of allmain variants of BP-2a whilst conserving the native structuralarchitecture of the selected domain. Interestingly, our results showthat the ability of the domains to elicit protective immunity was notdependent on the present of internal isopeptide bonds.

The cross protective immune response of the fusion proteins is offundamental importance in the development of a vaccine, since itdecreases the risk of generating escape mutants and enables thegeneration of a protective immune response against genetically differentGBS strains.

TABLE 1  Primers used in the experiments described herein SEQ ID PrimersSequence (5'-3') NO Gene amplified 515-D1 forCACCATGGAAGAAGCAAAAACTACTGAC 228 fragment coding for the 515-D1 revTCATTAATCAGCCAAGATAGAACCATC 229 domain 1 (30-162aa) of BP-2a 515 variant 515-D2 for CACCATGGATGGTTCTATCTTGGCTGAT 230fragment coding for the 515-D2 rev TCATTATTCAATTGTTGGGTTGTTGCC 231domain 2 (158-338aa) of BP- 2a 515 variant 515-D3 forCACCATGGGCAACAACCCAACAATTGAA 232 fragment coding for the 515-D3 revTCATTAAGCTTTTTCTGCATCTGTTGC 233 domain 3 (332-499aa) of BP-2a 515 variant 515-D4 for CACCATGTTGGCAGGAGCTACCTTCCTT 234fragment coding for the 515-D4 rev TCATTAAGTAACCTTCTTATTGATAAC 235domain 4 (472-640aa) of BP- 2a 515 variant H36B-D1CACCATGGCTGAGATGGGAAATATCACT 236 fragment coding for the fordomain 1 (30-158aa) of BP- H36B-D1 TCATTAGTCAGCAAGAACTTTGTCACC 2372a H36B variant rev H36B-D2 CACCATGGGTGACAAAGTTCTTGCTGAC 238fragment coding for the for domain 2 (152-350aa) of BP- H36B-D2revTCATTATACTTTTTTACCTGGTTTGTTACC 239 2a H36B variant H36B-D3CTGTACTTCCAGGGCAACAAACCAGGTAAAAAAGTA 240 fragment coding for the fordomain 3 (343-493aa) of BP- H36B-D3AATTAAGTCGCGTTATTATGCACCTTGCAAGCGTTCTGT 241 2a H36B variant rev H36B-D4CACCATGACAGAACGCTTGCAAGGTGCA 242 fragment coding for the fordomain 4 (487-658aa) of BP- H36B-D4rev TCATTAAGTCACTTTTTTGTTTTCTAT 2432a H36B variant BP-2a-H36B GTTTGCGCATATGGCTGAGATGGGAAATATCACT 244gene coding for the full for length BP-2a H36B variant BP-2a-H36BGTGGAATCTCGAGAGTCACTTTTTTGTTTTCTAT 245 without the signal peptide Revand the LPXTG (SEQ ID NO: 272) motif BP-2a 515CTGTACTTCCAGGGCGAAGAAGCAAAAACTACTGACACA 246 gene coding for the fullLPXTG-for GTG length BP-2a 515 variant  BP-2a 515AATTAAGTCGCGTTATGTACCAATACCACCTGTTTGTGG 247 with LPXTG (SEQ ID NO: 272)LPXTG-rev AAT motif. 6XD3 FP- CTGTACTTCCAGGGCAATAATCCGACCATTGAAAATG 248gene coding for the fusion for protein 6XD3 6XD3 FP-AATTAAGTCGCGTTAAATCGGCGTCGGATCGTTACTGTT 249 rev LYS42ALACACGCTATTGTCATGCCTCGAACTGCATTTGACGGTTTT 250 gene coding for the mutatedfor ACT form of BP-2a 515 variant LYS42ALACATGACAATAGCGTGCAAGGTCACTGTGTCAGTAGTTTT 251 containing K42A rev TGCLYS83ALA GAAGCGGCGGAAATCGCAGGTGCTTACTTTGCTTTC 252gene coding for the mutated for form of BP-2a 515 variant LYS83ALAGATTTCCGCCGCTTCGCCTGAGCCAAAGTAAGTTTTAAG 253 containing K83A revCJB111-D1 CACCATGGACGACGCAACAACTGATACT 254 fragment coding for fordomain 1 (30-162aa)  CJB111-D1 TCATTATGAATCAGCCAAGATAGAACCGTT 255of BP-2a CJB111 rev variant CJB111-D2 CACCATGAACGGTTCTATCTTGGCTGATTCA256 fragment coding for for domain 2 (155-337aa)  CJB111-D2TCATTATTCTTCCGTTGGGTTATTACC 257 of BP-2a CJB111 rev variant CJB111-D3CACCATGGGTAATAACCCAACGGAAGAA 258 fragment coding for fordomain 3 (331-474aa)  CJB111-D3 TCATTAAGCTCCTGCCAAGCGTTCAGT 259of BP-2a CJB111 rev variant CJB111-D4 CACCATGACTGAACGCTTGGCAGGAGCT 260fragment coding for for domain 4 (468-639aa)  CJB111-D4TCATTAGGTTACTTTTTTGTTTTGAACTTG 261 of BP-2a CJB111 rev variant

TABLE 2 Data collection and refinement statistics of BP-2a-515(molecular replacement). One crystal was used to solve the structure.Values in parentheses are for the highest resolution shell. BP-2a-515(residues 190-640) Data collection Space group P2₁2₁2₁ Cell dimensionsa, b, c (Å) 63.7, 104.7, 159.3 α = β = γ (°) 90 Resolution (Å) 40-1.75(1.75-1.84) R_(merge) 0.099 (0.6) I/σI 14.9 (3.7) Completeness (%) 100(100) Redundancy 9.6 (9.7) Refinement Resolution (Å) 40-1.75 No.reflections 103, 7178 R_(work)/R_(free) 18.5/21.6 No. atoms Protein 7076Potassium ion 3 Water 895 B-factors Protein 32.3 Potassium ion 24.8Water 20.2 R.m.s. deviations Bond lengths (Å) 0.007 Bond angles (°)1.047

TABLE 3 Results of an active maternal mouse immunization/neonatal pupchallenge model to determine protection conferred by single domain ofGBS59 515 variant against group B streptococcus 515 strain. Protectionconferred by single domains of BP-2a 515 variant against GBS 515 strainassessed by active maternal mouse immunization/neonatal pup challengemodel. Protection values was calculated as [(% dead in control − % deadin vaccine)/% dead in control]*100. Statistical significance AntigenAlive/Treated Protection (%) (p value) D1-515 17/59 20 0.0235 D2-515 6/25 15 0.1310 D3-515 19/28 64 p < .0001 D4-515 38/60 58 p < .0001BP-2a-515 full length 42/60 66 p < .0001 BP-2a-515 ΔIB 28/38 71 p <.0001 PBS  4/39

TABLE 4 Protection conferred by single domains of BP-2a-515 alleleagainst GBS strain 515, assessed by active maternal mouseimmunization/neonatal pup challenge model. Protection values werecalculated as [(% sepsis in control − % sepsis in vaccine)/% sepsis incontrol]*100. Statistical significance Antigen Protected/TreatedProtection (%) (p value)* D1-515 19/59 24 0.0098 D2-515  7/25 20 0.0687D3-515 21/28 72 p < .0001 D4-515 42/60 67 p < .0001 full lengthBP-2a-515 44/60 70 p < .0001 PBS  4/39 NOTE. Groups of female micereceived 3 doses (on days 1, 21, and 35) of either 20 μg antigen orbuffer (PBS) combined with Freund's adjuvant. Mice were then mated, andtheir offspring were challenged with a GBS dose calculated to inducesepsis in 90% of the pups. *p value, by Fisher's exact test.

TABLE 5 Protection conferred by single domains of BP-2a H36B variantagainst GBS 515 strain assessed by active maternal mouseimmunization/neonatal pup challenge model. Protection values wascalculated as [(% dead in control − % dead in vaccine)/% dead incontrol]*100. Statistical significance Antigen Alive/Treated Protection(%) (p value) D1-H36B 49/60 9 0.1562 D2-H36B 31/48 28 0.0016 D3-H36B 2/40 94 p < .0001 D4-H36B 19/37 43 p < .0001 BP-2a-H36B full length10/47 77 p < .0001 PBS 53/59

TABLE 6 Neonatal protection conferred by single domains of BP-2a-H36Bagainst GBS strain 5401, expressing the H36B BP-2a variant, assessed byactive maternal mouse immunization/neonatal pup challenge model.Protection values were calculated as [(% sepsis in control − % sepsis invaccine)/% sepsis in control]*100. Statistical significance AntigenProtected/Treated Protection (%) (p value)* D1-H36B 15/60 1 0.52 D2-H36B 22/48 29 0.019  D3-H36B 38/40 93 p < .0001 D4-H36B 20/37 390.0025 full length 39/47 78 p < .0001 BP-2a-H36B PBS 14/59 NOTE. Groupsof female mice received 3 doses (on days 1, 21, and 35) of either 20 μgantigen or buffer (PBS) combined with Freund's adjuvant. Mice were thenmated, and their offspring were challenged with a GBS dose calculated toinduce sepsis in 90% of the pups. *p value, by Fisher's exact test.

TABLE 7 Protection conferred by single domains of BP-2a-CJB111 variantagainst GBS CJB111 strain, expressing the BP-2a CJB111 variant, assessedby active maternal mouse immunization/neonatal pup challenge model.Protection values were calculated as [(% sepsis in control − % sepsis invaccine)/% sepsis in control]*100. Statistical significance AntigenProtected/Treated Protection (%) (p value)* D1-CJB111  2/39 0 0.3619D2-CJB111 12/50 16 0.065  D3-CJB111 41/54 73 p < .0001  D4-CJB111 13/4620 0.0269 full length 19/40 42 p = 0.00015 BP-2a-CJB111 PBS  4/41 NOTE.Groups of female mice received 3 doses (on days 1, 21, and 35) of either20 μg antigen or buffer (PBS) combined with Freund's adjuvant. Mice werethen mated, and their offspring were challenged with a GBS dosecalculated to induce sepsis in 90% of the pups. *p value, by Fisher'sexact test.

TABLE 8 Protection by active maternal mouse immunization/neonatal pupchallenge model conferred by fusion proteins against a panel of GBSstrains expressing different BP-2a allelic variants. Protection valueswas calculated as [(% dead in control − % dead in vaccine)/% dead incontrol]*100. GBS Protection (%) challenge BP-2a Fusion Protein FusionProtein Fusion Protein strains allele 6XD3 4XD3Helix (I) 4XD3Helix (II)515 515 63 43 62 CJB111 CJB111 70 52 36 3050 2603 68 100 79 5401 H36B 6590 60

TABLE 9 Protection by active maternal mouse immunization/neonatal pupchallenge model conferred by fusion protein 6xD3 against a panel of GBSstrains expressing different BP-2a allelic variants. Protection valueswere calculated as [(% sepsis in control − % sepsis in vaccine)/% sepsisin control]*100. GBS challenge BP-2a antigen 6xD3 PBS Protection strainallele (protected/treated) (protected/treated) (%) 515 515 50/68 13/5065* CJB111 CJB111 38/48  7/30 73* 3050 2603 53/70 12/40 66* 5401 H36B22/30 11/40 63* DK21 DK21 29/38  6/29 70* CDC89 CJB110 26/40  6/26 55*NOTE. Groups of female mice received 3 doses (on days 1, 21, and 35) ofeither 20 μg antigen or buffer (PBS) combined with Freund's adjuvant.Mice were then mated, and their offspring were challenged with a GBSdose calculated to induce sepsis in 90% of the pups. *p value, p < .0001by Fisher's exact test.

TABLE 10 Protection by active maternal mouse immunization/neonatal pupchallenge model conferred by fusion proteins with and without tagsagainst a panel of GBS strains. Fusion Fusion GBS protein 6xD3 - protein6xD3 - chal- His tag native PBS lenge (protected/ % (protected/ %(protected/ % strain treated) survival treated) survival treated)survival 515  59/104 57 43/69 62 16/83  19 5401 37/58 64 53/64 83 2/50 4CJB111 33/80 41 36/70 51 9/54 17

LIST OF SEQUENCES

-   SEQ ID NO:1 (GBS59 2603)-   SEQ ID NO:2 (GBS59 515)-   SEQ ID NO:3 (GBS59 cjb111)-   SEQ ID NO:4 (GBS59 h36b)-   SEQ ID NO:5 (GBS59 CJB110)-   SEQ ID NO:6 (GBS59 DK21)-   SEQ ID NO:7 (GBS59 NEM316)-   SEQ ID NO:8 (D1 2603)-   SEQ ID NO:9 (D2 2603)-   SEQ ID NO:10 (D3 2603)-   SEQ ID NO:11 (D4 2603)-   SEQ ID NO:12 (D1 515)-   SEQ ID NO:13 (D2 515)-   SEQ ID NO:14 (D3 515)-   SEQ ID NO:15 (D4 515)-   SEQ ID NO:16 (cjb111 D1)-   SEQ ID NO:17 (cjb111 D2)-   SEQ ID NO:18 (cjb111 D3)-   SEQ ID NO:19 (cjb111 D4)-   SEQ ID NO:20 (h36b D1)-   SEQ ID NO:21 (h36b D2)-   SEQ ID NO:22 (h36b D3)-   SEQ ID NO:23 (h36b D4)-   SEQ ID NO:24 (CJB110 D1)-   SEQ ID NO:25 (CJB110 D2)-   SEQ ID NO:26 (CJB110 D3)-   SEQ ID NO:27 (CJB110 D4)-   SEQ ID NO:28 (DK21 D1)-   SEQ ID NO:29 (DK21 D2)-   SEQ ID NO:30 (DK21 D3)-   SEQ ID NO:31 (DK21 D4)-   SEQ ID NO:32 (D1 NEM316)-   SEQ ID NO:33 (D2 NEM316)-   SEQ ID NO:34 (D3 NEM316)-   SEQ ID NO:35 (D4 NEM316)-   SEQ ID NO:36 (2603 D3 sub-fragment)-   SEQ ID NO:37 (2603 D4H)-   SEQ ID NO:38 (515 D3 sub-fragment)-   SEQ ID NO:39 (515 D4H)-   SEQ ID NO:40 (cjb111 D3 sub-fragment)-   SEQ ID NO:41 (cjb111 D4H)-   SEQ ID NO:42 (h36b D3 sub-fragment)-   SEQ ID NO:43 (h36b D4H)-   SEQ ID NO:44 (CJB110 D3 sub-fragment)-   SEQ ID NO:45 (CJB110 D4H)-   SEQ ID NO:46 (DK21 D3 sub-fragment)-   SEQ ID NO:47 (DK21 D4H)-   SEQ ID NO:48 (NEM316 D3 sub-fragment)-   SEQ ID NO:49 (DK21 D4H)-   SEQ ID NO:50 (2603 D3+D4)-   SEQ ID NO:51 (2603 D3+D4H)-   SEQ ID NO:52 (2603 D2+D3+D4)-   SEQ ID NO:53 (2603 D2+D3+D4H)-   SEQ ID NO:54 (515 D3+D4)-   SEQ ID NO:55 (515 D3+D4H)-   SEQ ID NO:56 (515 D2+D3+D4)-   SEQ ID NO:57 (515 D2+D3+D4H)-   SEQ ID NO:58 (cjb111 D3+D4)-   SEQ ID NO:59 (cjb111 D3+D4H)-   SEQ ID NO:60 (cjb111 D2+D3+D4)-   SEQ ID NO:61 (cjb111 D2+D3+D4H)-   SEQ ID NO:62 (h36b D3+D4)-   SEQ ID NO:63 (h36b D3+D4H)-   SEQ ID NO:64 (h36b D2+D3+D4)-   SEQ ID NO:65 (h36b D2+D3+D4H)-   SEQ ID NO:66 (CJB110 D3+D4)-   SEQ ID NO:67 (CJB110 D3+D4H)-   SEQ ID NO:68 (CJB110 D2+D3+D4)-   SEQ ID NO:69 (CJB110 D2+D3+D4H)-   SEQ ID NO:70 (DK21 D3+D4)-   SEQ ID NO:71 (DK21 D3+D4H)-   SEQ ID NO:72 (DK21 D2+D3+D4)-   SEQ ID NO:73 (DK21 D2+D3+D4H)-   SEQ ID NO:74 (NEM316 D3+D4)-   SEQ ID NO:75 (NEM316 D3+D4H)-   SEQ ID NO:76 (NEM316 D2+D3+D4)-   SEQ ID NO:77 (NEM316 D2+D3+D4H)-   SEQ ID NO:78 (515 short fragment of D3)-   SEQ ID NO:79 (his tag)-   SEQ ID NO:80 (linker)-   SEQ ID NO:81 (linker)-   SEQ ID NO:82 (linker)-   SEQ ID NO:83 (Fusion E)-   SEQ ID NO:84 (Fusion F)-   SEQ ID NO:85 (Fusion G)-   SEQ ID NO:86 (Fusion H)-   SEQ ID NO:87 (Fusion I)-   SEQ ID NO:88 (encoding Fusion E)-   SEQ ID NO:89 (encoding Fusion F)-   SEQ ID NO:90 (encoding Fusion G)-   SEQ ID NO:91 (encoding Fusion H)-   SEQ ID NO:92 (encoding Fusion I)-   SEQ ID NO:93 (encoding Fusion E—E. Coli optimised)-   SEQ ID NO:94 (encoding Fusion F—E. Coli optimised)-   SEQ ID NO:95 (encoding Fusion G—E. Coli optimised)-   SEQ ID NO:96 (encoding Fusion H—E. Coli optimised)-   SEQ ID NO:97 (encoding Fusion I—E. Coli optimised)-   SEQ ID NO:98 (IC adjuvant)-   SEQ ID NO:99 (Polycationic peptide adjuvant)-   SEQ ID NO:100 (encoding GBS59 2603)-   SEQ ID NO:101 (encoding GBS59 515)-   SEQ ID NO:102 (encoding GBS59 cjb111)-   SEQ ID NO:103 (encoding GBS59 h36b)-   SEQ ID NO:104 (encoding GBS59 CJB110)-   SEQ ID NO:105 (encoding GBS59 DK21)-   SEQ ID NO:106 (encoding GBS59 NEM316)-   SEQ ID NO:107 (encoding 2603 D1)-   SEQ ID NO:108 (encoding 2603 D2)-   SEQ ID NO:109 (encoding 2603 D3)-   SEQ ID NO:110 (encoding 2603 D4)-   SEQ ID NO:111 (encoding 515 D1)-   SEQ ID NO:112 (encoding 515 D2)-   SEQ ID NO:113 (encoding 515 D3)-   SEQ ID NO:114 (encoding 515 D4)-   SEQ ID NO:115 (encoding cjb111 D1)-   SEQ ID NO:116 (encoding cjb111 D2)-   SEQ ID NO:117 (encoding cjb111 D3)-   SEQ ID NO:118 (encoding cjb111 D4)-   SEQ ID NO:119 (encoding h36b D1)-   SEQ ID NO:120 (encoding h36b D2)-   SEQ ID NO:121 (encoding h36b D3)-   SEQ ID NO:122 (encoding h36b D4)-   SEQ ID NO:123 (encoding CJB110 D1)-   SEQ ID NO:124 (encoding CJB110 D2)-   SEQ ID NO:125 (encoding CJB110 D3)-   SEQ ID NO:126 (encoding CJB110 D4)-   SEQ ID NO:127 (encoding DK21 D1)-   SEQ ID NO:128 (encoding DK21 D2)-   SEQ ID NO:129 (encoding DK21 D3)-   SEQ ID NO:130 (encoding DK21 D4)-   SEQ ID NO:131 (encoding NEM316 D1)-   SEQ ID NO:132 (encoding NEM316 D2)-   SEQ ID NO:133 (encoding NEM316 D3)-   SEQ ID NO:134 (encoding NEM316 D4)-   SEQ ID NO:135 (encoding 2603 D3 sub-fragment)-   SEQ ID NO:136 (encoding 2603 D4H)-   SEQ ID NO:137 (encoding 515 D3 sub-fragment)-   SEQ ID NO:138 (encoding 515 D4H)-   SEQ ID NO:139 (encoding cjb111 D3 sub-fragment)-   SEQ ID NO:140 (encoding cjb111 D4H)-   SEQ ID NO:141 (encoding h36b D3 sub-fragment)-   SEQ ID NO:142 (encoding h36b D4H)-   SEQ ID NO:143 (encoding CJB110 D3 sub-fragment)-   SEQ ID NO:144 (encoding CJB110 D4H)-   SEQ ID NO:145 (encoding DK21 D3 sub-fragment)-   SEQ ID NO:146 (encoding DK21 D4H)-   SEQ ID NO:147 (encoding NEM316 D3 sub-fragment)-   SEQ ID NO:148 (encoding NEM316 D4H)-   SEQ ID NO:149 (encoding 2603 D3+D4)-   SEQ ID NO:150 (encoding 2603 D3+D4H)-   SEQ ID NO:151 (encoding 2603 D2+D3+D4)-   SEQ ID NO:152 (encoding 2603 D2+D3+D4H)-   SEQ ID NO:153 (encoding 515 D3+D4)-   SEQ ID NO:154 (encoding 515 D3+D4H)-   SEQ ID NO:155 (encoding 515 D2+D3+D4)-   SEQ ID NO:156 (encoding 515 D2+D3+D4H)-   SEQ ID NO:157 (encoding cjb111 D3+D4)-   SEQ ID NO:158 (encoding cjb111 D3+D4H)-   SEQ ID NO:159 (encoding cjb111 D2+D3+D4)-   SEQ ID NO:160 (encoding cjb111 D2+D3+D4H)-   SEQ ID NO:161 (encoding h36b D3+D4)-   SEQ ID NO:162 (encoding h36b D3+D4H)-   SEQ ID NO:163 (encoding h36b D2+D3+D4)-   SEQ ID NO:164 (encoding h36b D2+D3+D4H)-   SEQ ID NO:165 (encoding CJB110 D3+D4)-   SEQ ID NO:166 (encoding CJB110 D3+D4H)-   SEQ ID NO:167 (encoding CJB110 D2+D3+D4)-   SEQ ID NO:168 (encoding CJB110 D2+D3+D4H)-   SEQ ID NO:169 (encoding DK21 D3+D4)-   SEQ ID NO:170 (encoding DK21 D3+D4H)-   SEQ ID NO:171 (encoding DK21 D2+D3+D4)-   SEQ ID NO:172 (encoding DK21 D2+D3+D4H)-   SEQ ID NO:173 (encoding NEM316 D3+D4)-   SEQ ID NO:174 (encoding NEM D3+D4H)-   SEQ ID NO:175 (encoding NEM316 D2+D3+D4)-   SEQ ID NO:176 (encoding NEM316 D2+D3+D4H)-   SEQ ID NO:177 (GBS80 2603)-   SEQ ID NO:178 (GBS80 2603 without leader)-   SEQ ID NO:179 (GBS80 2603 without transmembrane/cytoplasmic region)-   SEQ ID NO:180 (GBS80 2603 without transmembrane/cytoplasmic region    and cell wall anchor)-   SEQ ID NO:181 (GBS80 2603 without extracellular domain)-   SEQ ID NO:182 (N-terminal immunogenic fragment of GBS80 2603)-   SEQ ID NO:183 (GBS67 2603)-   SEQ ID NO:184 (GBS67 2603 without transmembrane region)-   SEQ ID NO:185 (GBS67 2603 without transmembrane and cell wall anchor    motif)-   SEQ ID NO:186 (N-terminal fragment of GBS67 2603)-   SEQ ID NO:187 (N-terminal fragment of GBS67 2603)-   SEQ ID NO:188 (GBS67 h36b)-   SEQ ID NO:189 (N-terminal fragment of GBS67 h36b)-   SEQ ID NO:190 (N-terminal fragment of GBS67 h36b)-   SEQ ID NO:191 (GBS67 CJB111)-   SEQ ID NO:192 (N-terminal fragment of GBS67 CJB111)-   SEQ ID NO:193 (N-terminal fragment of GBS67 CJB111)-   SEQ ID NO:194 (GBS67 515)-   SEQ ID NO:195 (N-terminal fragment of GBS67 515)-   SEQ ID NO:196 (N-terminal fragment of GBS67 515)-   SEQ ID NO:197 (GBS67 NEM316)-   SEQ ID NO:198 (N-terminal fragment of GBS67 NEM316)-   SEQ ID NO:199 (N-terminal fragment of GBS67 NEM316)-   SEQ ID NO:200 (GBS67 DK21)-   SEQ ID NO:201 (N-terminal fragment of GBS67 DK21)-   SEQ ID NO:202 (N-terminal fragment of GBS67 DK21)-   SEQ ID NO:203 (GBS67 CJB110)-   SEQ ID NO:204 (N-terminal fragment of GBS67 CJB110)-   SEQ ID NO:205 (N-terminal fragment of GBS67 CJB110)-   SEQ ID NO:206 (GBS1523 COH1)-   SEQ ID NO:207 (GBS1523 COH1 without signal sequence region)-   SEQ ID NO:208 (GBS1523 COH1 with mutation at position 41)-   SEQ ID NO:209 (GBS80-GBS1523 hybrid)-   SEQ ID NO:210 (GBS80-GBS1523 hybrid)-   SEQ ID NO:211 (GBS80-GBS1523 hybrid)-   SEQ ID NO:212 (GBS80-GBS1523 hybrid)-   SEQ ID NO:213 (GBS104 2603)-   SEQ ID NO:214 (GBS1524)-   SEQ ID NO:215 (GBS3 2603)-   SEQ ID NO:216 (GBS3 2603 without signal sequence region)-   SEQ ID NO:217 (GBS3 2603 coiled coil and proline-rich segments)-   SEQ ID NO:218 (GBS3 2603 signal sequence and coiled coil)-   SEQ ID NO:219 (GBS3 2603 coiled coil segment)-   SEQ ID NO:220 (GBS3 2603 signal sequence, coiled coil and proline    rich segment)-   SEQ ID NO:221 (GBS3 515)-   SEQ ID NO:222 (GBS3 cjb111)-   SEQ ID NO:223 (GBS3 coh1)-   SEQ ID NO:224 (SAN1485 coh1)-   SEQ ID NO:225 (GBS147 2603)-   SEQ ID NO:226 (GBS328 2603)-   SEQ ID NO:227 (GBS84 2603)-   SEQ ID NO:228-261 (Primers)-   SEQ ID NO:262 (4H11/B7-VH DNA sequence)-   SEQ ID NO:263 (4H11/B7-VH amino acid sequence)-   SEQ ID NO:264 (4H11/B7-VLk DNA sequence)-   SEQ ID NO:265 (4H11/B7-VLk amino acid sequence)-   SEQ ID NO:266 (17C4/A3-VH DNA sequence)-   SEQ ID NO:267 (17C4/A3-VH amino acid sequence)-   SEQ ID NO:268 (17C4/A3-VLk DNA sequence)-   SEQ ID NO:269 (17C4/A3-VLk amino acid sequence)-   SEQ ID NO:270 (epitope of D3 bound by 4H11/B7 and 17C4/A3)-   SEQ ID NO:271 (RrgB)

REFERENCES

-   [1] Rosini et al, Molecular Microbiology, 2006, 61(1): 126-141-   [2] Margarit et al, Journal of Infectious Diseases, 2009, 199:    108-115-   [3] Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453.-   [4] Rice et al (2000) Trends Genet 16:276-277.-   [5] Bodanszky (1993) Principles of Peptide Synthesis (ISBN:    0387564314).-   [6] Fields et al. (1997) Meth Enzymol 289: Solid-Phase Peptide    Synthesis. ISBN: 0121821900.-   [7] Chan & White (2000) Fmoc Solid Phase Peptide Synthesis. ISBN:    0199637245.-   [8] Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413.-   [9] Ibba (1996) Biotechnol Genet Eng Rev 13:197-216.-   [10] U.S. Pat. No. 5,707,829-   [11] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN:    030644867X. Plenum.-   [12] WO90/14837.-   [13] WO90/14837.-   [14] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.-   [15] Podda (2001) Vaccine 19: 2673-2680.-   [16] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell    & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).-   [17] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan.-   [18] Allison & Byars (1992) Res Immunol 143:519-25.-   [19] Hariharan et al. (1995) Cancer Res 55:3486-9.-   [20] US-2007/014805.-   [21] WO95/11700.-   [22] U.S. Pat. No. 6,080,725.-   [23] WO2006/113373.-   [24] WO2005/097181.-   [25] U.S. Pat. No. 5,057,540.-   [26] WO96/33739.-   [27] EP-A-0109942.-   [28] WO96/11711.-   [29] WO00/07621.-   [30] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271.-   [31] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews    32:321-338.-   [32] Niikura et al. (2002) Virology 293:273-280.-   [33] Lenz et al. (2001) J Immunol 166:5346-5355.-   [34] Pinto et al. (2003) J Infect Dis 188:327-338.-   [35] Gerber et al. (2001) J Virol 75:4752-4760.-   [36] WO03/024480.-   [37] WO03/024481.-   [38] Gluck et al. (2002) Vaccine 20:B10-B16.-   [39] EP-A-0689454.-   [40] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.-   [41] Evans et al. (2003) Expert Rev Vaccines 2:219-229.-   [42] Meraldi et al (2003) Vaccine 21:2485-2491.-   [43] Pajak et al (2003) Vaccine 21:836-842.-   [44] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400.-   [45] WO02/26757.-   [46] WO99/62923.-   [47] Krieg (2003) Nature Medicine 9:831-835.-   [48] McCluskie et al. (2002) FEMS Immunology and Medical    Microbiology 32:179-185.-   [49] WO98/40100.-   [50] U.S. Pat. No. 6,207,646.-   [51] U.S. Pat. No. 6,239,116.-   [52] U.S. Pat. No. 6,429,199.-   [53] Kandimalla et al. (2003) Biochemical Society Transactions 31    (part 3):654-658.-   [54] Blackwell et al. (2003) J Immunol 170:4061-4068.-   [55] Krieg (2002) Trends Immunol 23:64-65.-   [56] WO01/95935.-   [57] Kandimalla et al. (2003) BBRC 306:948-953.-   [58] Bhagat et al (2003) BBRC 300:853-861.-   [59] WO03/035836.-   [60] Schellack et al. (2006) Vaccine 24:5461-72.-   [61] WO95/17211.-   [62] WO98/42375.-   [63] Beignon et al. (2002) Infect Immun 70:3012-3019.-   [64] Pizza et al (2001) Vaccine 19:2534-2541.-   [65] Pizza et al. (2000) Int J Med Microbiol 290:455-461.-   [66] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313.-   [67] Ryan et al (1999) Infect Immun 67:6270-6280.-   [68] Partidos et al. (1999) Immunol Lett 67:209-216.-   [69] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293.-   [70] Pine et al. (2002) J Control Release 85:263-270.-   [71] Tebbey et al. (2000) Vaccine 18:2723-34.-   [72] Domenighini et al. (1995) Mol Microbiol 15:1165-1167.-   [73] WO99/40936.-   [74] WO99/44636.-   [75] Singh et all (2001) J Cont Release 70:267-276.-   [76] WO99/27960.-   [77] U.S. Pat. No. 6,090,4060.-   [78] U.S. Pat. No. 5,916,588.-   [79] EP-A-0626169.-   [80] WO99/52549.-   [81] WO01/21207.-   [82] WO01/21152.-   [83] Andrianov et al (1998) Biomaterials 19:109-115.-   [84] Payne et al. (1998) Adv Drug Delivery Review 31:185-196.-   [85] Stanley (2002) Clin Exp Dermatol 27:571-577.-   [86] Jones (2003) Curr Opin Investig Drugs 4:214-218.-   [87] WO99/11241.-   [88] WO94/00153.-   [89] WO98/57659.-   [90] European patent applications 0835318, 0735898 and 0761231.-   [91] Donnelly et al (1997) Annu Rev Immunol 15:617-648.-   [92] Strugnell et al (1997) Immunol Cell Biol 75(4):364-369.-   [93] Cui (2005) Adv Genet 54:257-89.-   [94] Robinson & Torres (1997) Seminars in Immunol 9:271-283.-   [95] Brunham et al. (2000) J Infect Dis 181 Suppl 3:S538-43.-   [96] Svanholm et al (2000) Scand J Immunol 51(4):345-53.-   [97] DNA Vaccination—Genetic Vaccination (1998) eds. Koprowski et al    (ISBN 3540633928).-   [98] Gene Vaccination: Theory and Practice (1998) ed. Raz (ISBN    3540644288).-   [99] Findeis et al., Trends Biotechnol. (1993) 11:202-   [100] Chiou et at (1994) Gene Therapeutics: Methods And Applications    Of Direct Gene Transfer. ed. Wolff-   [101] Wu et al., J. Biol. Chem. (1988) 263:621-   [102] Wu et al., J. Biol. Chem. (1994) 269:542-   [103] Zenke et at, Proc. Natl. Acad. Sci. (USA) (1990) 87:3655-   [104] Wu et al., J. Biol. Chem. (1991) 266:338-   [105] Jolly, Cancer Gene Therapy (1994) 1:51-   [106] Kimura, Human Gene Therapy (1994) 5:845-   [107] Connelly, Human Gene Therapy (1995) 1:185-   [108] Kaplitt, Nature Genetics (1994) 6:148-   [109] WO 90/07936.-   [110] WO 94/03622.-   [111] WO 93/25698.-   [112] WO 93/25234.-   [113] U.S. Pat. No. 5,219,740.-   [114] WO 93/11230.-   [115] WO 93/10218.-   [116] U.S. Pat. No. 4,777,127.-   [117] GB Patent No. 2,200,651.-   [118] EP-A-0345242.-   [119] WO 91/02805.-   [120] WO 94/12649.-   [121] WO 93/03769.-   [122] WO 93/19191.-   [123] WO 94/28938.-   [124] WO 95/11984.-   [125] WO 95/00655.-   [126] Curiel, Hum. Gene Ther. (1992) 3:147-   [127] Wu, J. Biol. Chem. (1989) 264:16985-   [128] U.S. Pat. No. 5,814,482.-   [129] WO 95/07994.-   [130] WO 96/17072.-   [131] WO 95/30763.-   [132] WO 97/42338.-   [133] WO 90/11092.-   [134] U.S. Pat. No. 5,580,859-   [135] U.S. Pat. No. 5,422,120-   [136] WO 95/13796.-   [137] WO 94/23697.-   [138] WO 91/14445.-   [139] EP-0524968.-   [140] Philip, Mol. Cell Biol. (1994) 14:2411-   [141] Woffendin, Proc. Natl. Acad. Sci. (1994) 91:11581-   [142] U.S. Pat. No. 5,206,152.-   [143] WO 92/11033.-   [144] U.S. Pat. No. 5,149,655.-   [145] Zwijnenburg et al (2001) J Infect Dis 183:1143-6.-   [146] WO2009/016515.-   [147] Tettelin et al, Proc. Natl. Acad. Sci. U.S.A. 99 (19),    12391-12396 (2002)-   [148] Tettelin et al, Proc. Natl. Acad. Sci. U.S.A. 102 (39),    13950-13955 (2005)-   [149] WO09/101403-   [150] WO2006/130328-   [151] Lewis et al. (2004) PNAS USA 101:11123-8.-   [152] WO2006/050341.-   [153] Paoletti et al (1992) J Clin Invest 89:203-9-   [154] WO96/40795-   [155] Wessels et al. (1989) Infect Immun 57:1089-94.-   [156] WO2006/082527.-   [157] Paoletti et al (1990) J Biol Chem 265:18278-83.-   [158] Wessels et al. (1990) J Clin Invest 86:1428-33.-   [159] Paoletti et al. (1992) Infect Immun 60:4009-14.-   [160] Wessels et al. (1987) Proc Natl Acad Sci USA 84:9170-4.-   [161] Wang et al (2003) Vaccine 21:1112-7.-   [162] Wessels et al. (1993) Infect Immun 61:4760-6-   [163] Wessels et al. (1995) J Infect Dis 171:879-84.-   [164] Baker et al. (2004) J Infect Dis 189:1103-12.-   [165] U.S. Pat. No. 4,356,170.-   [166] Paoletti & Kasper (2003) Expert Opin Biol Ther 3:975-84.-   [167] WO2005/000346-   [168] Anonymous (January 2002) Research Disclosure, 453077.-   [169] Anderson (1983) Infect Immun 39(1):233-238.-   [170] Anderson et al. (1985) J Clin Invest 76(1):52-59.-   [171] EP-A-0372501.-   [172] EP-A-0378881.-   [173] EP-A-0427347.-   [174] WO93/17712.-   [175] WO94/03208.-   [176] WO98/58668.-   [177] EP-A-0471177.-   [178] WO91/01146.-   [179] Falugi et al. (2001) Eur J Immunol 31:3816-3824.-   [180] Baraldo et al. (2004) Infect Immun 72(8):4884-7.-   [181] EP-A-0594610.-   [182] Ruan et al. (1990) J Immunol 145:3379-3384.-   [183] WO00/56360.-   [184] WO01/72337.-   [185] WO00/61761.-   [186] WO00/33882-   [187] WO99/42130.-   [188] U.S. Pat. No. 4,761,283.-   [189] U.S. Pat. No. 4,356,170.-   [190] U.S. Pat. No. 4,882,317.-   [191] U.S. Pat. No. 4,695,624.-   [192] Mol Immunol. , 1985, 22, 907-919-   [193] EP-A-0208375.-   [194] Bethell G. S. et al., J. Biol. Chem., 1979, 254, 2572-4-   [195] Hearn M. T. W., J. Chromatogr., 1981, 218, 509-18-   [196] WO00/10599.-   [197] Gever et al., Med. Microbiol. Immunol, 165: 171-288 (1979).-   [198] U.S. Pat. No. 4,057,685.-   [199] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700.-   [200] U.S. Pat. No. 4,459,286.-   [201] U.S. Pat. No. 4,965,338.-   [202] U.S. Pat. No. 4,663,160.-   [203] WO2007/000343.-   [204] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN:    0-7216-9688-0.-   [205] Rappuoli et al (1991) TIBTECH 9:232-238.-   [206] Harper et al. (2004) Lancet 364(9447):1757-65.-   [207] Brandt et al. (2006) J Antimicrob Chemother. 58(6):1291-4.    Epub 2006 Oct. 26-   [208] Winter et al., (1991) Nature 349:293-99-   [209] U.S. Pat. No. 4,816,567.-   [210] Inbar et al., (1972) Proc. Natl. Acad. Sci. U.S.A. 69:2659-62.-   [211] Ehrlich et al., (1980) Biochem 19:4091-96.-   [212] Huston et al., (1988) Proc. Natl. Acad. Sci. U.S.A.    85:5897-83.-   [213] Pack et al., (1992) Biochem 31, 1579-84.-   [214] Cumber et al., (1992) J. Immunology 149B, 120-26.-   [215] Riechmann et al., (1988) Nature 332, 323-27.-   [216] Verhoeyan et al., (1988) Science 239, 1534-36.-   [217] GB 2,276,169.-   [218] Gennaro (2000) Remington: The Science and Practice of    Pharmacy. 20th edition, ISBN: 0683306472.-   [219] Methods In Enzymology (S. Colowick and N. Kaplan, eds.,    Academic Press, Inc.)-   [220] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications)

[221] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition (Cold Spring Harbor Laboratory Press).

-   [222] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed.,    CRC Press, 1997)-   [223] Ausubel et al. (eds) (2002) Short protocols in molecular    biology, 5th edition (Current Protocols).-   [224] Molecular Biology Techniques: An Intensive Laboratory Course,    (Ream et al., eds., 1998, Academic Press)-   [225] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton &    Graham eds., 1997, Springer Verlag)-   [226] Geysen et al (1984) PNAS USA 81:3998-4002.-   [227] Carter (1994) Methods Mol Biol 36:207-23.-   [228] Jameson, B A et al 1988, CABIOS 4(1):181-186.-   [229] Raddrizzani & Hammer (2000) Brief Bioinfonn 1(2):179-89.-   [230] Bublil et al. (2007) Proteins 68(1):294-304.-   [231] De Lalla et al. (1999) J. Immunol. 163:1725-29.-   [232] Kwok et al. (2001) Trends Immunol 22:583-88.-   [233] Brusic et al. (1998) Bioinformatics 14(2):121-30-   [234] Meister et al. (1995) Vaccine 13(6):581-91.-   [235] Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610.-   [236] Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7.-   [237] Feller & de la Cruz (1991) Nature 349(6311):720-1.-   [238] Hopp (1993) Peptide Research 6:183-190.-   [239] Welling et al. (1985) FEBS Lett. 188:215-218.-   [240] Davenport et al. (1995) Immunogenetics 42:392-297.-   [241] Tsurui & Takahashi (2007) J Pharmacol Sci. 105(4):299-316.-   [242] Tong et al. (2007) Brief Bioinform. 8(2):96-108.-   [243] Schirle et al. (2001) J Immunol Methods. 257(1-2):1-16.-   [244] Chen et al. (2007) Amino Acids 33(3):423-8.-   [245] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30-   [246] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.-   [247] Altschul (1997) Nucleic Acids Res. 1997 Sep. 1;    25(17):3389-402.-   [248] Proteins. 1995 November; 23(3):318-26.-   [249] Klock et al, (2008), Proteins, 71:982-994-   [250] Maione et al (2005), Science, 309: 148-150-   [251] Krissinel E., and Hendrick, K. (2007): J. Mol. Biol. 372:    774-797-   [252] Spraggon G. et al (2010) PLoS One 5, e10919

The invention claimed is:
 1. A method of raising, in a mammal, an immuneresponse against a Group B Streptococcus (GBS) strain expressingBackbone Protein-2a (BP-2a), compromising administering to the mammal aneffective amount of a polypeptide compromising the amino acid sequenceof SEQ ID NO:83.
 2. The method of claim 1 where said immune response isan antibody response.
 3. The method of claim 1 where said immuneresponse is a cell-mediated immune response.
 4. The method of claim 1where said immune response is cross-protective against GBS infectioncaused by GBS strains expressing different Backbone Protein-2a (BP-2a)allelic variants.
 5. The method of claim 1, where said polypeptidecomprises the amino acid sequence of SEQ ID NO:84.
 6. The method ofclaim 1, wherein said polypeptide comprises the amino acid sequence ofSEQ ID NO:85.
 7. The method of claim 1, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:86.
 8. The method ofclaim 1, wherein said polypeptide comprises the amino acid sequence ofSEQ ID NO:87.